Melt processed viral nanoparticle constructs

ABSTRACT

A construct includes a melt processed blend of a biodegradable polymer and viral nanoparticles. The viral nanoparticles are encapsulated within a biodegradable polymer matrix of the biodegradable polymer. The viral nanoparticles include a plurality of immunogenic peptides conjugated to the viral nanoparticles. The construct upon administration to a subject providing sustained release of the viral nanoparticles and conjugated immunogenic peptides from the matrix to the subject. The released viral nanoparticles and conjugated immunogenic peptides eliciting an immunogenic response in the subject.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application No.63/150,976, filed Feb. 18, 2021, and is a Continuation-in-Part of16/347,503, filed May 3, 2019, which claims priority from 62/417,000,filed Nov. 3, 2016, the subject matter of which are incorporated hereinby reference in their entirety.

GOVERNMENT FUNDING

This invention was made with government support under 1333651 awarded byThe National Science Foundation. The government has certain rights inthe invention.

p SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 18, 2022, isnamed CWR026164USCIPSEQUENCELISTING and is 8,438 bytes in size.

BACKGROUND

Biodegradable polymeric devices have been designed for drug delivery.Among the different classes of biodegradable polymers, the thermoplasticaliphatic poly(esters), such as poly(lactide) (PLA), poly(glycolide)(PGA), and especially the copolymer of lactide and glycolide referred toas poly(lactide-co-glycolide) (PLGA) have generated interest because oftheir excellent biocompatibility, biodegradability, and mechanicalstrength. These polymers are easy to formulate into various devices forcarrying a variety of drug classes, such as vaccines, peptides,proteins, and micromolecules, and have been approved by the UnitedStates Food and Drug Administration (FDA) for drug delivery.

Viral nanoparticles (VNPs) are a class of protein-based nanoparticlesthat have been extensively studied for immunology, biomedical, andagricultural applications. VNPs can consist of native or modified viralcapsid proteins encapsidating the viral genome, or self-assembled capsidproteins that are non-infectious, also termed virus-like particles(VLPs). The proteinaceous nature of VNPs makes them inherently morebiocompatible than synthetic nanoparticles derived from metals orpolymers. The precise self-assembly of VNPs yields monodisperse sizes,overcoming heterogeneity and lack of reproducibility often seen withsynthetic nanoparticles. The size range of VNPs is 20-500 nm, whichpromotes uptake by antigen presenting cells (APCs) and induction of animmune response. Furthermore, the surface of VNPs can be modified bycovalent coupling or genetic engineering to display multiple epitopes ina regular array to direct an immune response against a non-viral target.Five FDA approved VNP vaccines are currently on the market, with severalmore in clinical trials, further bolstering interest in developing newVNP delivery systems for immunology, drug delivery, and agriculture.

SUMMARY

Embodiments described herein relate to a melt processed viralnanoparticle construct that includes a degradable polymer matrix and aplurality of viral nanoparticles encapsulated within the degradablepolymer matrix. The nanoparticle construct can upon delivery and/oradministration to a site of interest provide a sustained and/orcontrolled release of the viral nanoparticles (e.g., virus or virus-likeparticles) to the site. The nanoparticle construct can also serve as asubstrate for the incorporation and/or attachment of at least one cargoagent and/or bioactive agent.

Advantageously, the melt processed nanoparticle construct can be formedwithout solvent (i.e., solvent-free or solventless), and the viralnanoparticles upon release from the degradable polymer matrix can havethe same or substantially similar structural and biochemicalcharacteristics as the viral nanoparticles prior to melt processing.

In some embodiments, where the nanoparticle construct is used fortherapeutic applications, the site of interest can be a cell or tissueof a subject. In other embodiments, where the nanoparticle construct isused for agricultural applications, the site of interest can be a plantpropagation material, a plant, part of a plant and/or plant organ.

In some embodiments, the melt processed nanoparticle construct can beprovided in a shape (e.g., plurality of microparticles or microneedles)that can be readily delivered to a subject to provide controlled and/orsustained release of the viral nanoparticles as well as cargo moleculesand/or bioactive agents coupled to and/or loaded on the construct tocells and/or tissue of a subject. The melt processed nanoparticleconstruct can be administered, injected, or implanted in a minimallyinvasive fashion in a subject in need thereof to treat diseases (e.g.,cancer) and/or disorder in the subject.

In some embodiments, the degradable polymer matrix can include a meltproces sable degradable polymer material that is biocompatible and, upondegradation, produces substantially non-toxic products. The viralnanoparticles can have a release profile from the degradable polymermatrix at least partially defined by the degradation of the degradablepolymer material under environmental and/or physiological conditions.

In other embodiments, the degradable polymer material can be meltprocessed at a Peclet number of about 5 to about 25. The degradablepolymer material can also have a melt temperature below a degradationtemperature of the viral nanoparticles.

In some embodiments, the degradable polymer material includespoly(lactic-co-glycolic acid) (PLGA) or a copolymer thereof. The polymermatrix can also include at least one porogen, such as polyethyleneglycol.

In some embodiments, the viral nanoparticle is a bacteriophage or plantvirus or plant virus-like particle. The bacteriophage virus orvirus-like particle can be Qβ-phage, AP 205-phage, GA-phage, fr-phage,or M2 phage. The plant virus or virus-like particle used to form thenanoparticle construct can be a plant picornavirus or a filamentousplant virus or virus like particle. The plant virus or virus-likeparticle can be of the Secoaviridoe genus or Alphafexiviridae family.For example, the plant virus or virus-like particle can be a cowpeamosaic virus-like particle or potato virus X virus-like particle. Inother embodiments, the plant virus particle or virus like particle canbe a rod-shaped virus particle. The rod-shaped virus can be a tobaccomosaic virus.

Other embodiments described herein relate to a viral nanoparticleconstruct that includes a melt processed blend of a biodegradablepolymer and viral nanoparticles. The viral nanoparticles can beencapsulated within a biodegradable polymer matrix of the biodegradablepolymer. The viral nanoparticles can include a plurality of immunogenicpeptides conjugated to the viral nanoparticles. The construct uponadministration to a subject can provide sustained release of the viralnanoparticles and conjugated immunogenic peptides from the matrix to thesubject. The released viral nanoparticles and conjugated immunogenicpeptides can elicit an immunogenic response in the subject to theconjugated immunogenic peptides.

In some embodiments, the conjugated immunogenic peptides upon releasefrom the polymer matrix can elicit substantially the same or enhancedimmunogenic response as immunogenic peptides conjugated to viralnanoparticles not encapsulated within the biodegradable polymer.

In some embodiments, the plurality of immunogenic peptides areconjugated to outer surfaces of the viral nanoparticles with linkers.The linkers can be a heterobifunctional linker.

In some embodiments, the viral nanoparticles are substantially uniformlydispersed in the polymer matrix.

In other embodiments, the biodegradable polymer material has a melttemperature below the degradation temperature of the viralnanoparticles. The biodegradable polymer material can includepoly(lactic-co-glycolic acid) (PLGA) or a copolymer thereof andoptionally at least one porogen, such as polyethylene glycol (PEG).

In some embodiments, the viral nanoparticles can include bacteriophages,plant viruses, and/or plant virus-like particles.

In some embodiments, the viral nanoparticles can have a release profilefrom the biodegradable polymer matrix at least partially defined by thedegradation of the biodegradable polymer material under physiologicalconditions.

In some embodiments, the construct can include up to about 25% by weightof the viral nanoparticles.

In other embodiments, the melt processing of biodegradable polymer andthe viral nanoparticles is at a Peclet number of about 5 to about 25.

In some embodiments, the construct can be configured as a plurality ofmicroparticles or microneedles.

Other embodiments describe herein relate to a single dose vaccineimplant that includes a biodegradable polymer and a plurality of viralnanoparticles melt processed with and encapsulated within abiodegradable polymer matrix that includes the biodegradable polymer.The viral nanoparticles can include a plurality of exogenous immunogenicpeptides conjugated to the viral nanoparticles. The implant uponadministration to a subject can provide sustained release of the viralnanoparticles and conjugated immunogenic peptides from the matrix to thesubject. The released viral nanoparticles and conjugated immunogenicpeptides can elicit an immunogenic response in the subject to theconjugated immunogenic peptides.

In some embodiments, the conjugated immunogenic peptides upon releasefrom the polymer matrix can elicit substantially the same or enhancedimmunogenic response as immunogenic peptides conjugated to viralnanoparticles not encapsulated within the biodegradable polymer.

In other embodiments, the plurality of immunogenic peptides can beconjugated to outer surfaces of the viral nanoparticles withheterobifunctional linkers.

In some embodiments, the viral nanoparticles are substantially uniformlydispersed in the polymer matrix.

In some embodiments, the biodegradable polymer material has melttemperature below the degradation temperature of the viral nanoparticlesand immunogenic peptide. The biodegradable polymer material can includepoly(lactic-co-glycolic acid) (PLGA) or a copolymer thereof andoptionally at least one porogen.

In some embodiments, the viral nanoparticles can include bacteriophages,plant viruses, and/or plant virus-like particles.

In some embodiments, the viral nanoparticles can have a release profilefrom the biodegradable polymer matrix at least partially defined by thedegradation of the biodegradable polymer material under physiologicalconditions.

In some embodiments, the implant can include up to about 25% by weightof the viral nanoparticles.

In some embodiments, the implant can be configured as a plurality ofmicroparticles or microneedles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(A-B) illustrate A) structures of PLGA and Qβ. B) A schematicdiagram of the syringe-die melt-encapsulation device showing theinternal structure of device and resulting cylindrical extrudates.

FIGS. 2(A-F) illustrate (A) FPLC chromatogram, (B) DLS histogram, and(C) TEM image of Qβ showing the typical Gaussian peak on thechromatogram and expected sizes in the DLS and TEM results before meltencapsulation. (D) FPLC chromatogram, (E) DLS histogram, and (F) TEMimage of recovered Qβ after melt-encapsulation with PLGA. The peak at 10mL in the FPLC chromatogram corresponded to aggregated species elutingat the void volume, which also appear as the larger peak in the DLShistogram. The TEM result, along with the FPLC and DLS result, indicatedthat a large proportion of the particles remain intact and are of thecorrect size.

FIGS. 3(A-F) illustrate EDS spectrum sulfur K-series emission signal (SK series) map of (A) 1 wt % Qβ, (B) 5 wt % Qβ, and (C) 10 wt % Qβ loadedPLGA material cross-sections indicating good dispersion of Qβ within thepolymeric matrix. Full-scale SEM images of (D) 1 wt % Qβ, (E) 5 wt % Qβ,and (F) 10 wt % Qβ loaded PLGA material cross-sections.

FIGS. 4(A-B) illustrate (A) FPLC chromatogram and (B) DLS histogram ofmelt-pressed and recovered Qβ indicating an increase in aggregatedspecies eluting at 10 mL in the chromatogram and an increase in the sizeof aggregated species centered at R_(h)=71.3 nm in the DLS histogram.

FIGS. 5(A-H) illustrate FPLC chromatograms of recovered Qβ samplessubjected to shear rates of (A) 0.1 s⁻¹, (B) 1 s⁻¹, (C) 10 s⁻¹, and (D.)50 s⁻¹. DLS plots of recovered Qβ samples subjected to shear rates of(E.) 0.1 s⁻¹, (F.) 1 s⁻¹, (G.) 10 s⁻¹, and (H.) 50 s⁻¹. Both FPLC andDLS results indicate that low shear rates, from 0.1 to 1 s⁻¹ resulted inan increase in aggregated species. Higher shear rates, up to 10 s⁻¹,dispersed aggregated species due to the higher shear forces applied.Shear rates exceeding 10 s⁻¹ induced particle break-up as evidenced bythe appearance of a large peak at 22.5 mL on the FPLC chromatogram.

FIGS. 6(A-B) illustrate (A) Plot of the mass average normalized radiusversus applied shear rate (top axis) and Peclet number (bottom axis).Plotting the aggregation behavior versus the shear rate and Pecletnumber allows for the determination of ideal processing parameters toprevent excessive aggregation and particle break-up of Qβ in PLGA andother polymeric systems during melt-processing (B) Applied energy to theparticles versus total particle disulfide energy analysis indicating thethermal and shear energy approached the level of disulfide energy in theQβ particles at 25 and 50 s⁻¹ shear rates, resulting in the observedparticle break-up.

FIGS. 7(A-B) illustrate release profiles of (A) 1 wt % Qβ loaded PLGAsamples with 10 wt % PEG 8K and PEG 20K additives and (B) 1, 5, and 10wt % loaded Qβ loaded PLGA. Reported as the average and standarddeviation of 3 samples.

FIGS. 8(A-C) illustrate (A) Immunization and bleeding schedule of miceimplanted with 0.5 cm of 10 wt % Qβ loaded PLGA and mice immunized via 3subcutaneous injections of 50 μg Qβ. (B) End-point titers of anti-Qβ IgGindicating the implanted PLGA/Qβ devices immunize as effectively asrepeated Qβ administration and (C) IgG subtype percentages of miceimmunized via subcutaneous injection and device implantation, whichindicate similar immune response via the same IgG subtype generationbetween mice immunized via injection and implantation. The arrowindicates a challenge with 50 μg Qβ for all mice and the IgG subtypeswere measured using sera collected on day 65. Titers and subtypepercentages are reported as the average and standard deviation ofmeasurements from 5 mice.

FIGS. 9(A-B) illustrate (A) SEM micrographs of PLGA microneedle arraysprepared via melt molding. The images were collected at 50× (left) and45× (right) magnification using a 10 kV accelerating voltage. (B) Forceversus displacement curves for the microneedle samples normalized as theforce per single needle. The maximum strength was defined as the firstplateau.

FIGS. 10(A-C) illustrate (A) FPLC chromatogram and (B) DLS histogram,and (C) TEM of Qβ recovered from the microneedle array.

FIG. 11 illustrates ELISA response from wild type, melt processed, andmicroneedle reprocessed Qβ. The absorbance at 405 nm, indicative of theantibody binding to Qβ, was normalized to the wild type Qβ value toyield a percent antibody recognition. The results are reported as theaverage and standard deviation from results using sera from 2 immunizedmice with sera for each sample read in triplicate.

FIGS. 12(A-B) illustrate (A) FPLC chromatogram and (B) DLS histogram ofQβ conjugated with Cy5. The 650 nm absorbance shown in the chromatogramwas indicative of the Cy5 dye.

FIG. 13 illustrates optical (left) and fluorescent (right) images ofporcine skin administered with PLGA microneedles laden with 10 wt %Qβ-Cy5. The administration site was visible in the optical image as thesquare indentation.

FIG. 14 illustrates amino acid sequences of wild type Qβ coat protein,Qβ coat protein fusion with P4, and Qβ coat protein fusion withCH401(Rat). The flexible linker was shown in blue, the P4 peptidesequence was shown in red, and the CH401(Rat) peptide sequence was shownin green. These amino acid sequences were cloned into an expressionplasmid and co-expressed with the wild type Qβ coat protein sequence(SEQ ID NOs: 7, 8, 9). The self-assembled particles displayed the P4 orCH401(Rat) peptide sequence on the particle surface.

FIGS. 15(A-C) illustrate (A) SDS-PAGE gel of (1) wild type Qβ, (2)Qβ-P4, and (3) Qβ-CH401(Rat). Wild type Qβ exhibited a single band at˜14 kDa indicating native Qβ coat protein. The appearance of a secondband for Qβ-P4 and Qβ-CH401(Rat) at -17 kDa was indicative of coatprotein fused to each peptide respectively (coat protein-P4=16.68 kDa,coat protein-CH401(Rat) =17.06 kDa). (B) MALDI-TOF spectrum of Qβ-P4exhibiting wild type coat protein at 14.17 kDa and the coat protein-P4fusion at 16.70 kDa (14.25 and 16.68 kDa calculated mass respectively).(C) MALDI-TOF spectrum of Qβ-CH401(Rat) exhibiting wild type coatprotein at 14.16 kDa and the coat protein-CH401(Rat) fusion at 16.96 kDa(14.25 and 17.06 kDa calculated mass respectively).

FIGS. 16(A-F) illustrate (A) FPLC chromatogram, (B) DLS histogram and(C) TEM micrograph of purified Qβ-P4 particles. (D) FPLC chromatogram,(E) DLS histogram, and (F) TEM micrograph of purified Qβ-CH401(Rat)particles.

FIG. 17 illustrates vaccination and sera collection scheme for treatmentgroups. 5 groups of 5 mice each were immunized via subcutaneousinjection of either 50 μg of wild type Qβ, Qβ-P4, or CH401(Rat) or 2 μgof free P4 or CH401(Rat) peptide. Serum was collected prior toinjections.

FIGS. 18(A-B) illustrate (A) P4 specific ELISA response for miceimmunized with Qβ, Qβ-P4, and P4 from sera collected on day 0, 14, 28,and 42. (B) CH401(Rat) specific ELISA response for mice immunized withQβ, Qβ-CH401(Rat), and CH401(Rat) from sera collected on day 0, 14, 28and 42. All results were reported as the average and standard deviationof measurements of sera from 5 mice.

FIG. 19 illustrates Qβ specific ELISA response for mice immunized withQβ, Qβ-P4, and Qβ-CH401(Rat) from sera collected on day 0, 14, 28, and42. All results were reported as the average and standard deviation ofmeasurements of sera from 5 mice.

FIGS. 20(A-C) illustrate (A) FPLC chromatogram, (B) DLS histogram, and(C) TEM micrograph of wild type Qβ melt processed at 10 wt % with PLGA.

FIGS. 21(A-F) illustrate (A) FPLC chromatogram, (B) DLS histogram, and(C) TEM micrograph of Qβ-P4 melt processed at 10 wt % with PLGA. (D)FPLC chromatogram, (E) DLS histogram, and (F) TEM micrograph ofQβ-CH401(Rat) melt processed at 10 wt % with PLGA.

FIG. 22 illustrates in vitro release profile of viral nanoparticles fromPLGA materials prepared via melt processing with 10 wt % of wild typeQβ, Qβ-P4, or Qβ-CH401(Rat). All results were reported as the averageand standard deviation of the release from 3 samples.

FIGS. 23(A-F) illustrate (A) FPLC chromatogram, (B) DLS histogram, and(C) TEM image of CPMV showing the typical Gaussian peak on thechromatogram and expected sizes in the DLS and TEM results. (D) FPLCchromatogram, (E) DLS histogram, and (F) TEM image of recovered CPMVafter melt processing with PLGA/15%PEG8000. The change in relativeintensities of the 280 and 260 nm absorbance in the melt processed FPLCchromatogram was due to loss of viral RNA after melt processing.

FIGS. 24(A-B) illustrate Agarose gel results for CPMV, CPMV recoveredvia organic extraction, and CPMV recovered via 24 hour aqueous release.(A) UV image of the gel showing RNA stained with 1% ethidium bromide and(B) optical image of the gel showing protein stained with Coomassie.

FIGS. 25(A-D) illustrate (A) SEM image of CPMV/PLGA/PEG8000microparticles collected at 500× magnification and 1.0 kV acceleratingvoltage. (B) Frequency histogram of microparticle diameter determinedfrom microparticle SEM images. Due to the range of round to ellipticalshapes exhibited by the particles, the diameter was defined as thelongest distance across a particle. The histogram was determined from150 particle measurements from 2 SEM images. (C) DLS histogram ofmicroparticles suspended in phosphate buffered saline. (D) Confocalimage of microparticles containing 5 wt % PLGA-FPI749 with particlesshown in green (λ_(ex)=635 nm, λ_(em)=700-800 nm).

FIGS. 26(A-B) illustrate Agarose gel results for CPMV and CPMV recoveredvia 24 hour aqueous release from microparticles. (A) UV image of the gelshowing RNA stained with 1% ethidium bromide and optical image of thegel showing protein stained with Coomassie. (B) DLS histogram of CPMVreleased from microparticles.

FIG. 27 illustrates in vitro release profile of CPMV released fromrod-shaped melt processed samples and CPMV released from cryo-milledmicroparticle samples. Reported as the average and standard deviation of3 samples.

FIGS. 28(A-B) illustrate (A) SEM image of PLGA/PEG8000 microneedlearrays collected at 30× (left) and 95× (right) magnification and 1.0 kVaccelerating voltage. (B) Force versus displacement curves for themicroneedle samples normalized as the force per single needle. Themaximum strength was defined as the first plateau of the force and thecurves are representative of 3 individual samples.

FIGS. 29(A-C) illustrate (A) DLS histogram of CPMV extracted from themicroneedle array. (B) Agarose gel results for CPMV and CPMV recoveredvia extraction from the microneedle array with the UV image of the gelshowing RNA stained with 1% ethidium bromide on the left and the opticalimage of the gel showing protein stained with Coomassie on the right.(C) TEM image of extracted CPMV from the microneedle array. Theirregular white signal arose due to the polymer background in thesample.

FIG. 30 illustrates ELISA response from wild type, melt processed, andmicroneedle reprocessed CPMV. The absorbance at 405 nm, indicative ofthe antibody binding to CPMV, was normalized to the wild type CPMV valueto yield a percent antibody recognition. The results are reported as theaverage and standard deviation (n=3). *p<0.05 determined via a twotailed t-test with equal variance assumed.

FIGS. 31(A-C) illustrate (A) Schematic of the injection schedule forOVCA treatment. Doses of 30, 100, and 500 μg were injected 4 times on aweekly schedule, denoted as ‘CPMV Inject’. A single dosage of eitherPLGA/PEG8000 microparticles or CPMV/PLGA/PEG8000 microparticles wereinjected at day 7, denoted as ‘Microparticle Inject’. (B) Totalluminescence from the luciferase reporter gene in the ID8-Debf29/Vegf-Aovarian cancer cells. The luminescence value was representative of tumorgrowth. Reported as the average and standard deviation of 5 mice foreach group. (C) Abdominal circumference measurement for each treatmentgroup, representative of fluid retention and tumor growth in theintraperitoneal space. Reported as the average and standard deviation of5 mice for each group.

FIG. 32 illustrates images of luminescence from the luciferase reporterin the ID8-Debf29/Vegf-A ovarian cancer cells for treatment groups onday 57.

FIG. 33 illustrates ELISA results for anti-CPMV IgG from sera collectedon day 46 for mice treated with 4 weekly injections of 100 μg CPMV(CPMV), mice treated with a single injection of PLGA/PEG8000microparticles (PLGA), and mice treated with a single injection of CPMVloaded PLGA/PEG8000 microparticles (PLGA/CPMV). Three dilution factorsof 500, 2500, and 12500 times dilution were used for the assay. Theresults are reported as the average and standard deviation of 2 mice foreach treatment group.

FIGS. 34 illustrates a plot showing total luminescence from theluciferase reporter gene in the ID8-Debf29/Vegf-A ovarian cancer cellsof mice injected with single dosage of either PLGA/PEG8000microparticles or CPMV/PLGA/PEG8000 microparticles coadministered withsoluble CPMV boost.

FIGS. 35(A-H) illustrate the synthesis, characterization, andimmunogenicity of the HPV-QP conjugates. (A) Schematic representation ofthe synthesis of four HPV-QP conjugates differing in the linkerstructure and epitope orientation (SEQ ID NOs: 11-14). (B) SDS-PAGEanalysis of the Qβ and HPV-QP particles: (1) Qβ, (2) GPSL-N-expo-HPV-Qβ,(3) GPSL-C-expo-HPV-Qβ, (4) GGSG-N-expo-HPV-Qβ, and (5)GGSG-C-expo-HPV-Qβ. (C) FPLC chromatogram of GGSG-C-expo-HPV-Qβ. (D) TEMimage of GGSG-C-expo-HPV-QP (scale bar=100 nm). (E) DLS analysis ofGGSG-C-expo-HPV-Qβ. (F) Serum IgG titers against the HPV peptidefollowing immunization with the four HPV-Qβ conjugates or Qβ. (G) SerumIgG titers against Qβ following immunization with the four HPV-Qβconjugates or Qβ. (H) ELISA signals at 405 nm for mice vaccinated withdifferent antigens at different dilutions after the second boost. Micewere subcutaneously injected with 30 μg of each agent on day 0 (prime),14 (first boost), and 28 (second boost). Blood was collected 7 daysafter each injection (Data are means ±S.D. for n=5 mice per group).Asterisks show significance as determined by unpaired two-tailedstudent's t-test: ns, not significant p>0.05; * p<0.05; *** p<0.005.

FIGS. 36(A-D) illustrate the preparation and characterization ofmelt-extruded PLGA implants. (A) Equipment for the melt processing ofpolymer materials. (B) PLGA implants produced by melt extrusion. Thelong implant bar produced directly from the device is shown at the top,with the short sections ready for implantation shown underneath. Thephotograph shows a metric ruler. The white bars are plain PLGA+Qβ-HPV,and the green ones are FITC-PLGA +Cy5-Qβ. (C) SEM image showing across-section of the PLGA implant loaded with Qβ-HPV. (D) The EDXspectrum sulfur K-series emission signal (SK series) map of the PLGAimplant loaded with Qβ-HPV.

FIGS. 37(A-E) illustrate slow release of Qβ-HPV from the PLGA implant.(A) The release curve of the PLGA implants loaded with Qβ-HPV or Qβ,with the particles released into PBS at 37° C. (data are means ±standarddeviations, n=3). (B) SDS-PAGE analysis of (1) unmodified Qβ standardand Qβ-HPV released from the implant between (2) 0-15 days and (3) 16-30days. (C) TEM image of the released Qβ-HPV particles on day 30. (D) FPLCanalysis of the released Qβ-HPV particles on day 30. (E) DLS analysis ofthe released Qβ-HPV particles on day 30.

FIGS. 38(A-C) illustrate the release of Cy5-Qβ particles from PLGAimplants in vivo. (A) Fluorescence images of mice containing FITC-PLGAimplants loaded with Cy5-Q3, showing the Cy5 channel (top) and GFPchannel (bottom) at different time-points. Blue square shows implantlocation. Scale bar shows fluorescence intensity for all images in eachchannel. (B) Quantitative release profile (Cy5 channel) for theFITC-PLGA implants loaded with Cy5-Qβ (Data are means ±s.d. for n=4 miceper group). (C) Quantitative release profile (GFP channel) for theFITC-PLGA implants loaded with Cy5-Qβ (Data are means ±s.d. for n=4 miceper group).

FIGS. 39(A-D) illustrate single-dose vaccination using PLGA implantsloaded with HPV-Qβ. (A) Vaccination and bleeding schedule for mice withsubcutaneous PLGA implants or equivalent HPV-Qβ injections. (B) Serumtiters of HPV-specific IgG for mice vaccinated with three subcutaneousinjections of 30 μg HPV-Qβ or a single-dose PLGA implant loaded with 100i.ig HPV-Qβ, 500 μg HPV-Qβ, a mixture of 100 μg Qβ and 20 μg HPVpeptide, or 20 μg HPV peptide alone (Data are means ±s.d. for n=6 miceper group). Statistical significance was determined by unpairedtwo-tailed student's t-test: ns, not significant with p>0.05. (C)HPV-specific IgG subtype ratio for mice vaccinated with a single-dosePLGA implant containing 100 i.ig HPV-Qβ, or three subcutaneous doses of35 μg HPV-Qβ. Blood was collected on day 35 (Data are means ±s.d. forn=6 mice per group). (D) Serum (day 35) from three mice immunized withvarious vaccine formulations tested in duplicate for neutralizationagainst HPV16 pseudovirus at ID₆₀ (pseudovirus infectious dose thatinfects 60-70% of control cells). Infected cells (expressing GFP) wereidentified by flow cytometry (data are means ±standard errors based onthe relative percentage of infected cells in wells exposed to serumcompared to non-exposed controls).

FIGS. 40(A-D) illustrate the characterization of Qβ and HPV-Qβconjugates by TEM, FPLC, and DLS. (A) Qβ, (B) GPSL-N-expo-HPV-Qβ, (C)GPSL-C-expo-HPV-Qβ, and (D) GGSG-N-expo-HPV-Qβ.

FIGS. 41(A-B) illustrate ELISA signals at 405 nm for mice vaccinatedwith various antigens at different dilutions after (A) prime vaccinationand (B) the first boost (Data are means ±s.d. for n=5 mice per group).Asterisks show significance, as determined by unpaired two-tailedstudent's t-test: ns, not significant p >0.05; * p<0.05; ** p<0.01; ***p<0.005.

FIGS. 42(A-B) illustrate the vaccination with HPV-Qβ solutions. Theserum IgG antibody titer against (A) the HPV peptide and (B) Qβ for micevaccinated with 30 μg HPV-Qβ, a mixture of 5 μg HPV and 30 μg Qβ, or 5μg HPV peptide alone. Mice were subcutaneously injected with the agentson days 0 (prime), 14 (first boost), and 21 (second boost). Blood wascollected 7 days after each injection (n=6 for each group).

FIGS. 43(A-B) illustrate the characterization of Cy5-Qβ. (A) FPLCchromatogram of Cy5-Qβ. (B) SDS-PAGE analysis of Cy5-Qβ (lane 1) andunmodified Qβ (lane 2). The Coomassie Brilliant Blue channel is shown onthe left, and the blue fluorescence channel on the right; M =sizemarkers.

FIGS. 44(A-B) illustrates the immunization of mice with Qβ particles.(A) The serum titer of QP-specific IgG in mice vaccinated with threesubcutaneous injections of 30 μg HPV-Qβ or a single dose of 100 μgHPV-Qβ, 500 μg HPV-Qβ, a mixture of 100 μg Qβ and 20 i.ig HPV peptide,or 20 μg HPV peptide alone, in each case loaded on a subcutaneous PLGAimplant. (B) QP-specific IgG subtype ratio in mice vaccinated with asingle-dose PLGA implant containing 100 μg HPV-Qβ, or three doses of 35μg HPV-Qβ (Data are means ±s.d. for n=6 mice per group). Statisticalsignificance was determined by unpaired two-tailed student's t-test; ns,not significant with p>0.05.

FIGS. 45(A-D) illustrates the production and characterization of HPV16pseudovirus (PsHPV). A) Pseudovirus purification afterultracentrifugation. The red arrow indicates the band corresponding tothe intact pseudovirus particles. B) Different fractions (1-18) ofpseudovirus particles prepared by ultracentrifugation and separated by2% agarose gel electrophoresis. The presence of bands indicatespseudovirus DNA. C) SDS-PAGE analysis (14-20% gels) of the samefractions tested in B, showing resolved bands representing Ll (50-55kDa) and histone (15 kDa). Fractions 9-18 were considered pseudoviruspositive and were used for the neutralization assay. MW=molecularweight. D) Pooled fractions (9-18) titrated and analyzed by flowcytometry. The presence of GFPpositive cells indicates an infection.

FIGS. 46(A-C) illustrate the immunization with HPV-Qβ at different dosesand schedules. Mice were immunized with (i) single dose PLGA implantloaded with 100 μg HPV-Qβ (ii) single subcutaneous injection of 100mHPV-Qβ solution, and (iii) three subcutaneous injections of 30 μg HPV-Qβsolution. Six mice were studied per group. (A) Schedule for vaccinationand bleeding for all three vaccine formulations. Blood collection wasdone every two weeks. (B) Serum titers of HPV-specific IgG forvaccinated mice on day 14, day 28, day 42, day 56, and day 90. Thescattered points represent individual mouse and the horizontal linesrepresent the mean for each group. (C) Kinetic profile of HPV-specificIgG antibody titer for mice groups vaccinated with different vaccineformulation-100 μg of HPV-Qβ loaded implant, single subcutaneousinjection of 100 μg HPV-Qβ solution, and three subcutaneous injectionsof 30 μg HPV-Qβ solution. Error bars represent uncertainties (±1σ) foreach mice group.

DETAILED DESCRIPTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for describing particularembodiments only and is not intended to be limiting of the invention.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

As used in the description of the invention and the appended claims, thesingular forms “a,” “an,” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise. Inaddition, the recitations of numerical ranges by endpoints include allnumbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

The term “about” when referring to a measurable value such as an amount,a temporal duration, and the like, is meant to encompass variations of±20% or 110%, more preferably ±5%, even more preferably ±1%, and stillmore preferably ±0.1% from the specified value, as such variations areappropriate to perform the disclosed methods.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

The term “bioactive agent” can refer to any agent capable of promoting abiological effect, e.g., alters or modulates a biological function of aphysiological target substance. By “alters” or “modulates a biologicalfunction” herein is meant that the physiological target undergoes achange in either the quality or quantity of its biological activity;this includes increases or decreases in activity. Thus, bioactive agentsinclude a wide variety of drugs, including antagonists, for exampleenzyme inhibitors, and agonists, for example a transcription factorwhich results in an increase in the expression of a desirable geneproduct (although as will be appreciated by those in the art,antagonistic transcription factors may also be used), are all included.

In addition, a “bioactive agent” includes those agents capable of directtoxicity and/or capable of inducing toxicity towards healthy and/orunhealthy cells in the body. Also, the bioactive agent may be capable ofinducing and/or priming the immune system against potential pathogens. Anumber of mechanisms are possible including without limitation, (i) aradioisotope linked to a protein as is the case with a radiolabledprotein, (ii) an antibody linked to an enzyme that metabolizes asubstance, such as a produg, thus rendering it active in vivo, (iii) anantibody linked to a small molecule therapeutic agent, (iv) aradioisotope, (v) a carbohydrate, (vi) a lipid, (vii) a thermal ablationagent, (viii) a photosensitizing agent, and (ix) a vaccine agent.

The terms “biocompatible” and “biologically compatible” refer tomaterials that are, along with any metabolites or degradation productsthereof, generally non-toxic to the recipient, and do not cause anysignificant adverse effects to the recipient, at concentrationsresulting from the degradation of the administered materials. Generallyspeaking, biocompatible materials are materials that do not elicit asignificant inflammatory or immune response when administered to apatient.

The term “biodegradable polymer” generally refers to a polymer that willdegrade or erode by enzymatic action or hydrolysis under physiologicconditions to smaller units or chemical species that are capable ofbeing metabolized, eliminated, or excreted by the subject. Thedegradation time is a function of polymer composition, morphology, suchas porosity, particle dimensions, and environment.

The term “cargo molecule,” refers to a small organic or inorganicbioactive agent, such as a drug or imaging agent that can be associatedwith a virus nanoparticle in order to confer an additional function onthe virus nanoparticle.

The term “controlled release” refers to control of the rate and/orquantity of a virus nanoparticles, cargo molecules, and/or bioactiveagents delivered using the nanoconstructs described herein. Thecontrolled release can be continuous or discontinuous, and/or linear ornon-linear. This can be accomplished using one or more types of polymermaterials or compositions, drug loadings, inclusion of excipients ordegradation enhancers, or other modifiers, administered alone, incombination or sequentially to produce the desired effect.

The term “effective amount” refers to an amount of virus nanoparticles,cargo molecules, and/or bioactive agents that is sufficient to provide adesired effect. An effective amount in any individual case may bedetermined by one of ordinary skill in the art using routineexperimentation.

The term “imaging agent” can refer to a biological or chemical moietycapable being linked and/or conjugated directly or indirectly tonanoparticle constructs described herein and that may be used to detect,image, and/or monitor the presence and/or progression of a cell cycle,cell function/physiology, condition, pathological disorder and/ordisease.

The term “linker” or “linker molecule,” as used herein, refers to amolecule including linker region made up of a long hydrophilic carbonchain or hydrophilic polymer, and two or more attachment sites providedat the ends of the linker molecule that allow the linker to be reactedwith virus particles and/or attachment sites on a support surface.

The terms “matrix” and “polymer matrix” refer to a three-dimensionalnetwork of polymer materials or compounds. The polymer materials orcompounds are arranged in such a way as to permit the inclusion of othermaterials compounds inside the three dimensional network.

The term “subject” can be a human or non-human animal. Non-human animalsinclude, for example, livestock and pets, such as ovine, bovine,porcine, canine, feline and murine mammals, as well as reptiles, birdsand fish. Preferably, the subject is human.

Embodiments described herein relate to a melt processed viralnanoparticle construct that includes a degradable polymer matrix and aplurality of virus or virus-like particles encapsulated within thedegradable polymer matrix. The nanoparticle construct can upon deliveryand/or administration to a site of interest provide a sustained and/orcontrolled release of the virus or virus-like particles to the site. Thenanoparticle construct can also serve as a substrate for theincorporation and/or attachment of at least one cargo agent and/orbioactive agent.

Advantageously, the melt processed nanoparticle construct can be formedwithout solvent (i.e., solvent-free or solventless), and the virus orvirus-like particles upon release from the degradable polymer matrix canhave the same or substantially similar structural (e.g., size, shape,and morphology) and biochemical (e.g., immune response) characteristicsas the virus or virus-like particles prior to melt processing.

In some embodiments, where the nanoparticle construct is used fortherapeutic applications, the site of interest can be a cell or tissueof a subject. In other embodiments, where the nanoparticle construct isused for agricultural applications, the site of interest can be a plantpropagation material, a plant, part of a plant and/or plant organ.

In some embodiments, the nanoparticle construct can be provided in ashape (e.g., a plurality of microparticles or microneedles) that can bereadily delivered to a subject to provide controlled and/or sustainedrelease of the virus or virus-like particles as well as cargo moleculesand/or bioactive agents coupled to and/or loaded on the construct tocells and/or tissue of a subject. The nanoparticle construct can beadministered, injected, or implanted in a minimally invasive manner in asubject in need thereof to treat diseases (e.g., cancer) and/ordisorders in the subject.

The degradable polymer matrix can include or be made of a melt processable degradable polymer material. The melt processable degradablepolymer material can be, for example, hydrolytically degradable,biodegradable, thermally degradable, and/or photolytically degradable.

The degradable polymer material can also have a melt temperature thatallows the degradable polymer to be readily processed by, for example,melt extrusion, and below the degradation temperature of the virus orvirus-like particles. For example, the degradable polymer material canhave a melt temperature below about 120° C., about 110° C., about 100°C., about 90° C., about 80° C., or about 70° C. and be readily extrudedwithout the aid of solvents with the virus or virus-like particles toform the melt processed nanoparticle construct.

Degradable polymers can include, for example, certain polyesters,polyanhydrides, polyorthoesters, polyphosphazenes, polyphosphoesters,certain polyhydroxyacids, polypropylfumerates, polycaprolactones,polyamides, poly(amino acids), polyacetals, polyethers, biodegradablepolycyanoacrylates, biodegradable polyurethanes and polysaccharides. Forexample, biocompatible, biodegradable, or bioerodible polymers includepoly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lacticacid-co-glycolic acid)s (PLGAs), polyanhydrides, polyorthoesters,polyetheresters, polycaprolactones (PCL), polyesteramides, poly(butyricacid), poly(valeric acid), polyvinylpyrrolidone (PVP), polyvinyl alcohol(PVA), polyethylene glycol (PEG), block copolymers of PEG-PLA,PEG-PLA-PEG, PLA-PEG-PLA, PEG-PLGA, PEG-PLGA-PEG, PLGA-PEG-PLGA,PEG-PCL, PEG-PCL-PEG, PCL-PEG-PCL, copolymers of ethyleneglycol-propylene glycol-ethylene glycol (PEG-PPG-PEG, trade name ofPluronic or Poloxamer) and copolymers and blends of these polymers.

In certain embodiments, poly(lactic-co-glycolic acid) (PLGA) can be usedas the melt processable biodegradable polymer. Biodegradable polymersfabricated from PLGA have emerged as powerful potential carriers forsmall and large molecules of therapeutic importance as well as scaffoldsfor tissue engineering applications. This importance derives from: 1)Physiologic compatibility of PLGA and its hompolymers PGA and PLA, allof which have been established as safe in humans after 30 years invarious biomedical applications including drug delivery systems 2)Commercial availability of a variety of PLGA formulations for controlover the rate and duration of molecules released for optimalphysiological response(Visscher et al. J Biomed Mater Res 1985;19(3):349-65; Langer R, Folkman J. Nature 1976; 263(5580):797-800;Yamaguchi. J. Controlled Rel. 1993; 24(1-3):81-93.). 3) Biodegradabilityof PLGA materials, which provides for sustained release of theencapsulated molecules under physiologic conditions while degrading tonontoxic, low-molecular-weight products that are readily eliminated(Shive et al. Adv Drug Deliv Rev 1997; 28(1):5-24; Johansen et al. Eur JPharm Biopharm 2000; 50(1):129-46). 4) Control over its manufacturinginto nanoscale particles (<500 nm) for potential evasion of the immunephagocytic system or fabrication into microparticles on the length scaleof cells for targeted delivery of drugs or as antigen-presentingsystems(Eniola et al. J Control Release 2003; 87(1-3):15-22; Jain R A.Biomaterials 2000; 21(23):2475-90).

The degradable polymers described herein can have a variety of molecularweights. The polymers may, for example, have molecular weights of atleast about 5 kD, at least about 10 kD, at least about 20 kD, at leastabout 22 kD, at least about 30 kD, or at least about 50 kD.

The degradable polymers and derivatives thereof can be selected andadapted to have a desired degradation rate. Alternatively oradditionally, a degradation rate may be fine-tuned by associating ormixing other materials (e.g., non-degradable materials) with one or moreof degradable polymer material.

In general, a degradation rate as used herein can be dictated by thetime in which a material degrades a certain percentage (e.g., 50%) in acertain condition (e.g., in physiological conditions). In someembodiments, the degradation time of the nanoparticle construct or aportion of the nanoparticle construct as described herein can have awide range. In some embodiments, the degradation time may be greaterthan 1 minute, 5 minutes, 30 minutes, 1 hour, 2 hours, 5 hours, 12hours, 24 hours, 1.5 days, 2 days, 5 days, 7 days, 15 days, 30 days, 2months, 6 months, 1 year, 2 years, or even 5 years. In embodiments, thedegradation time may be about or less than 10 years, 5 years, 2 years, 1year, 6 months, 2 months, 30 days, 15 days, 7 days, 5 days, 2 days, 1.5days, 24 hours, 12 hours, 5 hours, 2 hours, 1 hour, 30 minutes or even 5minutes. The degradation time may be in a range of 12-24 hours, 1-6months, or 1-5 years. In some embodiments, the degradation time may bein a range of any two values above.

In other embodiments, the nanoparticle construct is designed to releasevirus or virus-like particles to be encapsulated or attached over aperiod of days to weeks. Factors that affect the duration of releaseinclude pH of the surrounding medium (higher rate of release at pH 5 andbelow due to acid catalyzed hydrolysis of PLGA) and polymer composition.Aliphatic polyesters differ in hydrophobicity and that in turn affectsthe degradation rate. Specifically, the hydrophobic poly (lactic acid)(PLA), more hydrophilic poly (glycolic acid) PGA and their copolymers,poly (lactide-co-glycolide) (PLGA) have various release rates. Thedegradation rate of these polymers, and often the corresponding drugrelease rate, can vary from days (PGA) to months (PLA) and is easilymanipulated by varying the ratio of PLA to PGA.

Optionally, the degradable polymer matrix can include one or moreporogens to enhance or accelerate the release of the virus or virus likeparticles from the melt processed nanoparticle construct. The porogencan be added during melt processing of the degradable polymer materialand virus or virus-like particles. The porogen can include any compoundthat will reserve a space within the degradable polymer matrix of thenanoparticle construct while the nanoparticle construct is being formed.

Porogens may be of any shape or size. A porogen may be spheroidal,cuboidal, rectangular, elonganted, tubular, fibrous, disc-shaped,platelet-shaped, polygonal, etc. In certain embodiments, the porogen isgranular with a diameter ranging from about 10 μm to about 800 μm (e.g.,about 25 μm to about 100 μm). In certain embodiments, a porogen iselongated, tubular, or fibrous.

The amount of porogens may vary in the formation of the nanoparticleconstruct and range from about 1% to about 80% by weight. Pores canprovide for easier degradation of the degradable polymer matrix andfacilitate release of the virus or virus-like particles from thedegradable polymer matrix. In some embodiments, a porogen isbiocompatible.

In some embodiments, a porogen may be a gas, liquid, or solid. Examplesof possible solid porogens include water soluble compounds. Exemplaryporogens include peptides and proteins (e.g., gelatin), carbohydrates,salts, sugar alcohols, natural polymers, synthetic polymers, and smallmolecules. In one example, the porogen can be polyethylene glycol, whichis a known porogen of PLGA materials.

The nanoparticle construct can be formed using a variety of differenttypes of viral nanoparticles. The term viral nanoparticle can refer tovirus or virus particles, which include a nucleic acid encoding thevirus, and virus-like particles, which do not include a nucleic acidencoding the virus. Viral nanoparticles are readily modifiable throughboth genetic engineering and chemical modification, with a well-ordered,multivalent display of functional groups on exterior as well as interiorsurfaces of viral nanoparticles. They are also highly economical asproduction can be scaled up using molecular farming or fermentation.

Viral nanoparticles encapsulated by the melt processable degradablepolymer matrix described herein can be categorized based on their sourceand structure. For example, viral nanoparticles from mammalian, avian,bacterial, or plant sources can be used. One advantage of using viralnanoparticles from plant sources is that they can be readily cultivatedand are unlikely to cause infection when used in vivo in a subject. Inaddition, viral nanoparticles having a helical, icosahedral, or prolatestructure can be used. Examples of helical viruses include tobaviruses,such as tobacco mosaic tobavirus and filamentous bacteriophages, e.g.,M13 and fd. A variety of helical viruses are described by Stubbs et al.,Adv. Exp. Med. Bio., 726, p. 631-658 (2012), the disclosure of which isincorporated herein by reference. Examples of icosahedral virusesinclude Qβ, P22 and other bacteriophages, HIV, herpesvirus, adenovirus,poliovirus, human papillomavirus, and picornaviruses, as well as variousplant viruses, such as cowpea mosaic virus, brome mosaic virus, cowpeachlorotic mottle virus, etc.

In some embodiments, the bacteriophage virus or virus-like particle canbe Qβ-phage, AP 205-phage, GA-phage, fr-phage, or M2 phage.

In other embodiments, the plant virus or virus-like particles can be anicosahedral plant virus or virus-like particle. Examples of icosahedralplant viruses include the virus families Geminiviridae, Luteoviridae,Bromoviridae, Phycodnaviridae, and Picornaviridae. In some embodiments,the icosahedral plant virus is from the family Picornaviridae. Plantpicornaviruses are relatively small, non-enveloped, positive-strandedRNA viruses with an icosahedral capsid. Plant picornaviruses have anumber of additional properties that distinguish them from otherpicornaviruses, and are categorized as the subfamily secoviridae. Insome embodiments, the virus particles are selected from the Comovirinaevirus subfamily. Examples of viruses from the Comovirinae subfamilyinclude Cowpea mosaic virus, Broad bean wilt virus 1, and Tobaccoringspot virus. In a further embodiment, the virus particles are fromthe Genus comovirus. An example of a comovirus is the cowpea mosaicvirus particles.

In other embodiments, the plant virus or virus like particle is afilamentous plant virus. Filamentous plant virus is a virus thatprimarily infects plants and has a non-enveloped filamentous structure.A filamentous structure is a long, thin virion that has a filament-likeor rod-like shape that is much longer than it is wide and therefore hasa high-aspect ratio. For example, Alphaflexiviridae have a length ofabout 470 to about 800 nm, and a diameter of about 12-13 nm.

In some embodiments, the filamentous plant virus belongs to a specificvirus family, genus, or species. For example, in some embodiments, thefilamentous plant virus belongs to the Alphaflexiviridae family. TheAlphaflexiviridae family includes the genus Allexivirus, Botrexvirus,Lolavirus, Mandarivirus, Potexvirus, and Sclerodamavirus. In someembodiments, the filamentous plant virus belongs to the genusPotexvirus. In further embodiments, the filamentous plant virus belongsto the Potato Virus X species.

In other embodiments, the plant virus or virus-like particle can bebased on a rod-shaped plant virus or virus like particle. A rod-shapedplant virus is a virus that primarily infects plants, is non-enveloped,and is shaped as a rigid helical rod with a helical symmetry. Rod shapedviruses also include a central hollow canal. Rod-shaped plant virusparticles are distinguished from filamentous plant virus particles as aresult of being inflexible, shorter, and thicker in diameter. Forexample, Virgaviridae have a length of about 200 to about 400 nm, and adiameter of about 15-25 nm. Virgaviridae have other characteristics,such as having a single-stranded RNA positive sense genome with a3′-tRNA like structure and no polyA tail, and coat proteins of 19-24kilodaltons.

In some embodiments, the rod-shaped plant virus belongs to a specificvirus family, genus, or species. For example, in some embodiments, therod-shaped plant virus belongs to the Virgaviridae family. TheVirgaviridae family includes the genus Furovirus, Hordevirus,Pecluvirus, Pomovirus, Tobamovirus, and Tobravirus. In some embodiments,the rod-shaped plant virus belongs to the genus Tobamovirus. In furtherembodiments, the rod-shaped plant virus belongs to the tobacco mosaicvirus species. The tobacco mosaic virus has a capsid made from 2130molecules of coat protein and one molecule of genomic single strand RNA6400 bases long. The coat protein self-assembles into the rod likehelical structure (16.3 proteins per helix turn) around the RNA whichforms a hairpin loop structure. The protein monomer consists of 158amino acids which are assembled into four main alpha-helices, which arejoined by a prominent loop proximal to the axis of the virion. Virionsare about 300 nm in length and about 18 nm in diameter. Negativelystained electron microphotographs show a distinct inner channel of about4 nm.

The virus or virus-like particles can be obtained according to variousmethods known to those skilled in the art. In embodiments where plantvirus particles are used, the virus particles can be obtained from theextract of a plant infected by the plant virus. For example, cowpeamosaic virus can be grown in black eyed pea plants, which can beinfected within 10 days of sowing seeds. Plants can be infected by, forexample, coating the leaves with a liquid containing the virus, and thenrubbing the leaves, preferably in the presence of an abrasive powderwhich wounds the leaf surface to allow penetration of the leaf andinfection of the plant. Within a week or two after infection, leaves areharvested and viral nanoparticles are extracted. In the case of cowpeamosaic virus, 100 mg of virus can be obtained from as few as 50 plants.Procedures for obtaining plant picornavirus particles using extractionof an infected plant are known to those skilled in the art. See WellinkJ., Meth Mol Biol, 8, 205-209 (1998). Procedures are also available forobtaining virus-like particles. Saunders et al., Virology, 393(2):329-37(2009). The disclosures of both of these references are incorporatedherein by reference.

The viral nanoparticles can be encapsulated within the degradablepolymer matrix by melt processing, such as melt encapsulation. In meltencapsulation, dry powders of degradable polymer material, virus orvirus-like paticles and other additives are mixed and then heated abovethe melt or glass transition of the polymer material but below thedegradation temperature of the virus or virus-line particles. Themixture can then be molded (e.g., compression molded and/orextrusion/injection molded) and cooled to a desired shape orconfiguration. The melt processing (e.g., melt encapsulation) and/orpost processing (e.g., extrusion) conditions of the mixture can becontrolled such that the concentrations of the materials in the mixtureare relatively consistent throughout and a melt processed nanoparticleconstruct is provided in which the viral nanoparticles can besubstantially uniformly dispersed within the degradable polymer matrixand the virus or virus-like particles upon release from the degradablepolymer matrix can have the same or substantially similar size, shape,and biochemical characteristics (e.g., immune response) as the virus orvirus-like particles prior to melt processing.

The conditions during melt processing can be defined or measured interms of a Peclet number. Advantageously, a Peclet number of about 5 toabout 25 is maintained during melt processing of degradable polymermaterial and the virus or virus-like particles. Maintaining the Pecletnumber between about 5 to about 25 during melt processing of thedegradable polymer material and the virus or virus-like particles allowsthe virus or virus-like particles to be uniformly dispersed within thedegradable polymer matrix without aggregation and maintain the virus orvirus-like particles' structural and biochemical integrity (e.g., immuneresponse).

The viral nanoparticles can be mixed or loaded with the degradablepolymer material at loading levels of about 1%, 5%, 20%, 25% or more toprovide a nanoparticle construct with viral nanoparticle loading levelsof about 1%, 5%, 20%, 25% or more. The loading level can influence therelease profile form the degradable polymer matrix of the nanoparticleconstruct. In some embodiments, increasing the loading level canincrease the amount of viral nanoparticles initially released from thenanoparticle construct.

In some embodiments, the melt processed nanoparticle constructs can beloaded with one or more cargo molecules and/or bioactive agents. Forexample, the cargo molecule and/or bioactive agent can be loaded bycovalently attaching the cargo molecule and/or bioactive agent to areactive molecule on a surface (interior or exterior) of one or morevirus or virus like particles prior to melt processing. In otherembodiments, the nanoparticle construct can be loaded with cargomolecules and/or bioactive agents by mixing the cargo molecules and/orbioactive agents with the viral nanoparticles and degradable polymermaterial during melt processing.

A variety of different types of cargo molecules and/or bioactive agentscan be loaded into or onto the nanoparticle constructs. Cargo moleculesand/or bioactive agents are generally relatively small organic orinorganic molecules. In some embodiments, the cargo molecules have amolecular weight ranging from about 50 to about 5000 daltons, with someembodiments being directed to cargo molecules having a weight rangingfrom about 50 to about 1000 daltons, or from about 100 to about 500daltons. Examples of cargo molecules are imaging agents and therapeuticagents, such as antitumor agents.

In some embodiments, the nanoparticle construct is modified to carry animaging agent. Examples of imaging agents include fluorescent compounds,radioactive isotopes, and MRI contrast agents. For example, in someembodiments, the imaging agent is a fluorescent molecule for fluorescentimaging. The imaging agent can be any material having a detectablephysical or chemical property. Such imaging agents have beenwell-developed in the field of fluorescent imaging, magnetic resonanceimaging, positive emission tomography, or immunoassays and, in general,most any imaging agent useful in such methods can be applied to thepresent invention. Thus, an imaging agent is any composition detectableby spectroscopic, photochemical, biochemical, immunochemical,electrical, optical or chemical means. Means of detecting imaging agentsare well known to those of skill in the art. Thus, for example, wherethe imaging agent is a radioactive compound, means for detection includea scintillation counter or photographic film as in autoradiography.Where the imaging agent includes a fluorescent label, it may be detectedby exciting the fluorochrome with the appropriate wavelength of lightand detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors, such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel.

Examples of imaging agents that can be used include magnetic beads(e.g., Dynabeads), fluorescent dyes (e.g., fluorescein isothiocyanate,AlexaFluor555, Texas red, rhodamine, and the like), radiolabels (e.g.,³H ¹⁴C, ³⁵S, ¹²⁵I, ¹²¹I, ¹¹²In, ^(99m)Tc), other imaging agents such asmicrobubbles (for ultrasound imaging), 18_(F) 11C ^(is)o, (for Positronemission tomography), ^(99m)TC, ¹¹¹In (for single photon emissiontomography), and chelated lanthanides such as terbium, gadolinium (e.g.,chelated gadolinium), and europium or iron (for magnetic resonanceimaging). The choice of imaging agent depending on sensitivity required,ease of conjugation with the compound, stability requirements, availableinstrumentation, and disposal provisions.

In some embodiments, the imaging agent is a magnetic resonance imagingagent. Disease detection using MRI is often difficult because areas ofdisease have similar signal intensity compared to surrounding healthytissue. In the case of magnetic resonance imaging, the imaging agent canalso be referred to as a contrast agent. Lanthanide elements are knownto be useful as contrast agents. The lanthanide chemical elementscomprises the fifteen metallic chemical elements with atomic numbers 57through 71, and include lanthanum, cerium, praseodymium, neodymium,promethium, samarium, europium, gadolinium, terbium, dysprosium,holmium, erbium, thulium, ytterbium, and lutetium. Preferred lanthanidesinclude europium, gadolinium, and terbium. In order to more readilyhandle these rare earth metals, the lanthanides are preferably chelated.In some embodiments, the lanthanide selected for use as a contrast agentis gadolinium, or more specifically gadolinium (III). Gadoliniumcontrast agents are generally chelated to facilitate attachment to thevirus particle. Examples of effective gadolinium chelating moleculesinclude 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA),diethylenetriaminopentacetate (DTPA),1,4,7,10-tetraazacyclododecane-1,4,7-triasacetic acid (DO3A),6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid (AAZTA), and4-carboxyamido-3,2-hydroxypyridinone (HOPA).

In other embodiments, the cargo molecule and/or bioactive agent can be atherapeutic agent. Examples of therapeutic agents include cardiovasculardrugs (e.g., antihypertensive drugs, antiarrhythmic agents, anddiuretics), neuropharmaceuticals (e.g., analgesics, anesthetics, andantipsychotics), gastrointestinal drugs (e.g., anti-ulcer drugs,antiemetics, and gastroprokinetic agents), respiratory tract agents(e.g., anthasthamtic or antiallergic drugs), antiinfective agents(antibiotics, antimycotics, and antiviral agents), endocrine-affectingdrugs (e.g., steroids, hormones, and contraceptives), anti-inflammatorydrugs, immunosuppressant drugs, and antitumor agents.

In some embodiments, the therapeutic agents used as cargo molecules aresmall molecule antitumor agents. One advantage of using antitumor agentsas cargo molecules is the ability of viral nanoparticles topreferentially associate with tumor cells. Examples of small moleculeantitumor agents include angiogenesis inhibitors such as angiostatinK1-3, DL-α-difluoromethyl-ornithine, endostatin, fumagillin, genistein,minocycline, staurosporine, and thalidomide; DNA intercalating orcross-linking agents such as bleomycin, carboplatin, carmustine,chlorambucil, cyclophosphamide, cisplatin, melphalan, mitoxantrone, andoxaliplatin; DNA synthesis inhibitors such as methotrexate,3-Amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin, cytosineβ-D-arabinofurano side, 5-Fluoro-5′-deoxyuridine, 5-Fluorouracil,gaciclovir, hydroxyurea, and mitomycin C; DNA-RNA transcriptionregulators such as actinomycin D, daunorubicin, doxorubicin,homoharringtonine, and idarubicin; enzyme inhibitors such asS(+)-camptothecin, curcumin, deguelin, 5,6-dichlorobenz-imidazole1-β-D-ribofuranoside, etoposine, formestane, fostriecin, hispidin,cyclocreatine, mevinolin, trichostatin A, tyrophostin AG 34, andtyrophostin AG 879, Gene Regulating agents such as5-aza-2′-deoxycitidine, 5-azacytidine, cholecalciferol,4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, alltrans-retinal, all trans retinoic acid, 9-cis-retinoic acid, retinol,tamoxifen, and troglitazone; Microtubule Inhibitors such as colchicine,dolostatin 15, nocodazole, paclitaxel, podophyllotoxin, rhizoxin,vinblastine, vincristine, vindesine, and vinorelbine; and various otherantitumor agents such as 17-(allylamino)-17-demethoxygeldanamycin,4-Amino-1,8-naphthalimide, apigenin, brefeldin A, cimetidine,dichloromethylene-diphosphonic acid, leuprolide,luteinizing-hormone-releasing hormone, pifithrin-.alpha., rapamycin,thapsigargin, and bikunin, and derivatives thereof.

The cargo molecules and/or bioactive agents can be conjugated to thevirus or virus-like particles and/or other materials (e.g., degradablepolymer material) of the melt processed nanoparticle constructs by anysuitable technique, with appropriate consideration of the need forpharmacokinetic stability and reduced overall toxicity to the patient.The term “conjugating” when made in reference to an agent and ananoparticle construct as used herein means covalently linking the agentto virus or virus like particles and/or other material of thenanoparticle construct subject to the limitation that the nature andsize of the agent and the site at which it is covalently linked to thevirus or virus like particles and/or other material of the nanoparticleconstruct do not interfere with the distribution of the virus or viruslike particles of the nanoparticle construct. The cargo molecule can belinked to the interior or the exterior of virus or virus like particlesand/or other material of the nanoparticle construct, while in someembodiments the cargo molecule is linked to both the interior and theexterior of the virus or virus like particles and/or nanoparticleconstruct. In some embodiments, where the cargo molecule is linked to avirus or virus like particle, the location of the cargo molecule on theinterior or exterior is governed by the amino acids of viral coatproteins that are selected as reactive sites.

Cargo molecules and/or bioactive agents can be coupled to the virus orvirus-like particles and/or other materials of the nanoparticleconstruct either directly or indirectly (e.g., via a binder group). Insome embodiments, the molecule and/or agent is directly attached to afunctional group capable of reacting with the agent and/or molecule. Forexample, viral coat proteins include lysines that have a free aminogroup that can be capable of reacting with a carbonyl-containing group,such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide). Viral coat proteinsalso contain glutamic and aspartic acids. The carboxylate groups ofthese amino acids also present attractive targets for functionalizationusing carbodiimide activated linker molecules; cysteines can also bepresent which facilitate chemical coupling via thiol-selective chemistry(e.g., maleimide-activated compounds). Further, viral coat proteinscontain tyrosines, which can be modified using diazonium couplingreactions. In addition, genetic modification can be applied to introduceany desired functional residue, including non-natural amino acids, e.g.,alkyne- or azide-functional groups. See Hermanson, G. T. BioconjugationTechniques. (Academic Press, 2008) and Pokorski, J. K. and N. F.Steinmetz, Mol Pharm 8(1): 29-43 (2011), the disclosures of which areincorporated herein by reference.

In other embodiments, a chemical binder group can be used. A bindergroup can serve to increase the chemical reactivity of a substituent oneither the agent or the virus or virus like particles and/or othermaterials of the nanoparticle construct, and thus increase the couplingefficiency. Binder chemistries can include maleimidyl binders, which canbe used to bind to thiol groups, isothiocyanate and succinimidyl (e.g.,N-hydroxysuccinimidyl (NHS)) binders, which can bind to free aminegroups, diazonium which can be used to bind to phenol, and amines, whichcan be used to bind with free acids such as carboxylate groups usingcarbodiimide activation. Useful functional groups are present on viralcoat proteins based on the particular amino acids present, andadditional groups can be designed into recombinant viral coat proteins.It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), can be employed as a binder group.Coupling can be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues.

Other types of binding chemistries are also available. For example,methods for conjugating polysaccharides to peptides are exemplified by,but not limited to coupling via alpha- or epsilon-amino groups toNaIO₄-activated oligosaccharide (Bocher et al., J. Immunol. Methods 27,191-202 (1997)), using squaric acid diester(1,2-diethoxycyclobutene-3,4-dione) as a coupling reagent (Tietze et al.Bioconjug Chem. 2:148-153 (1991)), coupling via a peptide binder whereinthe polysaccharide has a reducing terminal and is free of carboxylgroups (U.S. Pat. No. 5,342,770), and coupling with a synthetic peptidecarrier derived from human heat shock protein hsp65 (U.S. Pat. No.5,736,146). Further methods for conjugating polysaccharides, proteins,and lipids to plant virus peptides are described by U.S. Pat. No.7,666,624.

In some embodiments, the surface of the virus or virus-like particlescan be modified by attachment of something other than a cargo molecule.For example, the virus particle can be modified to include PEGylation,cell penetrating peptides, or targeting molecules. The nanoparticleconstruct can be modified either before loading the virus or virus-likeparticles with cargo molecules, after loading with cargo molecules,prior to melt processing, and/or after melt processing. Targetingmolecules can be attached to the outside of the virus or virus-likeparticles in order to guide the virus or virus like particles uponrelease from the nanoparticle construct to a particular target tissue,such as tumor tissues. Examples of targeting molecules include peptideligands (e.g., RGD, bombesin, or GE11), vitamins such as folic acid, andother tumor-homing proteins such as transferrin, as well as andantibodies such as Herceptin or any other antibody or antibody fragmentwith tumor-specific properties, and DNA-, RNA-, or PNA-based aptamersthat specifically bind to an antigen present on the target tissue, suchas a tumor antigen. Cell penetrating peptides can also be attached tothe outside of the virus or virus-like particles to encourageinternalization of the virus or virus like particles constructs. Cellpenetrating peptides are generally relatively short, amphipathicpeptides. Examples of cell penetrating peptides include TAT sequence orpolyArginine peptides.

In some embodiments, rather than covalent attachment, cargo moleculesand/or bioactive agents can also be loaded into or onto the nanoparticleconstructs in a non-covalent manner by associating them with nucleicacid present within the virus particles of the nanoparticle construct.While not intending to be bound by theory, it appears that the cargomolecule associates with the nucleic acid as a result of the affinity ofthe cargo molecule and/or bioactive agent for the nucleic acid. Affinityis the tendency of a compound to naturally associate with another object(e.g., a nucleic acid). Affinity is influenced by non-covalentintermolecular interactions between the compound and the object, such ashydrogen bonding, electrostatic interactions, hydrophobic interactions,and Van der Waals forces.

An example of cargo molecules having an affinity for the nucleic acidare cargo molecules having a positive charge. One skilled in the art canreadily determine whether a cargo molecule has affinity for the nucleicacid within a plant virus particle. For example, gel mobility shiftassays, oligonucleotide crosslinking assays, optical absorbance andfluorescence assays, calorimetric assays, and/or surface Plasmonresonance assays to determine the association and dissociation kineticsand affinities of cargo molecules for nucleic acids.

Furthermore, any drug or imaging agent exhibiting low affinity can bereadily modified with a small, positively charged tag or complementaryoligonucleotide to bind to nucleic acid within a virus particle. Forsome embodiments, the cargo molecules interact with nucleic acids in areversible manner, in order to facilitate release of the cargo moleculesin or to the target tissue.

In some embodiments, a targeting molecule can also be attached to thevirus or virus-like particles of the nanoparticle constructs. By“targeting molecule” herein is meant a molecule which serves to targetor direct the virus or virus-like particles released from thenanoparticle construct to a particular location, cell type, diseasedtissue, or association. In general, the targeting molecule is directedagainst an antigenic site. Thus, for example, antibodies, cell surfacereceptor ligands and hormones, lipids, sugars and dextrans, alcohols,bile acids, fatty acids, amino acids, peptides and nucleic acids may allbe attached to localize or target the virus or virus-like particles to aparticular site. In some embodiments, the targeting molecule allowstargeting of the virus or virus-like particles to a particular tissue orthe surface of a cell.

In some embodiments, the targeting molecule is a peptide. For example,chemotactic peptides have been used to image tissue injury andinflammation, particularly by bacterial infection; see WO 97/14443,hereby expressly incorporated by reference in its entirety. Anotherexample are peptides specific to fibrin or vascular cell adhesionmolecules to direct the imaging probe to sites of inflammation, such asan atherosclerotic plaque. In other embodiments, the targeting moleculeis an antibody. The term “antibody” includes antibody fragments, as areknown in the art, including Fab Fab₂, single chain antibodies (Fv forexample), chimeric antibodies, etc., either produced by the modificationof whole antibodies or those synthesized de novo using recombinant DNAtechnologies. In further embodiments, the antibody targeting moieties ofthe invention are humanized antibodies or human antibodies. Humanizedforms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.

In some embodiments, the antibody is directed against a cell-surfacemarker on a cancer cell; that is, the antigenic site is a cell surfacemolecule. As is known in the art, there are a wide variety of antibodiesand antibody fragments known to be differentially expressed on tumorcells, including, but not limited to, HER2. Examples of physiologicallyrelevant carbohydrates may be used as cell-surface markers include, butare not limited to, antibodies against markers for breast cancer (CA15-3, CA 549, CA 27.29), mucin-like carcinoma associated antigen (MCA),ovarian cancer (CA125), pancreatic cancer (DE-PAN-2), and colorectal andpancreatic cancer (CA 19, CA 50, CA242).

In some embodiments, the targeting molecule is all or a portion (e.g., abinding portion) of a ligand for a cell surface receptor. Suitableligands include, but are not limited to, all or a functional portion ofthe ligands that bind to a cell surface receptor selected from the groupconsisting of insulin receptor (insulin), insulin-like growth factorreceptor (including both IGF-1 and IGF-2), growth hormone receptor,glucose transporters (particularly GLUT 4 receptor), transferrinreceptor (transferrin), epidermal growth factor receptor (EGF), lowdensity lipoprotein receptor, high density lipoprotein receptor, leptinreceptor, estrogen receptor (estrogen); interleukin receptors includingIL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12,IL-13, IL-15, and IL-17 receptors, human growth hormone receptor, VEGFreceptor (VEGF), PDGF receptor (PDGF), transforming growth factorreceptor (including TGF-alpha. and TGF-beta), EPO receptor (EPO), TPOreceptor (TPO), ciliary neurotrophic factor receptor, prolactinreceptor, and T-cell receptors. Receptor ligands include ligands thatbind to receptors such as cell surface receptors, which includehormones, lipids, proteins, glycoproteins, signal transducers, growthfactors, cytokines, and others.

The nanoparticle construct can be injectable and/or implantable, and canbe in the form of, for example, a membrane, sponge, gel, solid scaffold,spun fiber, woven or unwoven mesh, nanoparticles, microparticles,microneedle array or any other desirable configuration. The nanoparticleconstruct can be used in a variety of biomedical applications, includingtissue engineering, drug discovery applications, and regenerativemedicine and cancer therapy.

In one embodiment, as illustrated in FIG. 9, the nanoparticle constructcan be in the form of a microneedle array that includes a plurality ofmicroprotrusion or microprojections that can be used to penetrate theskin or other biological membranes. In general, the microprojections canhave a height of at least about 50 μm, at least about 100 μm, at leastabout 150 μm, at least about 200 μm, at least about 250 μm, or at leastabout 300 μm m. In general, it is also preferred that themicroprojections have a height of no more than about 1 mm, no more thanabout 500 μm, no more than about 300 μm, or in some cases no more thanabout 200 μm or 150 μm. The microprojections may have an aspect ratio ofat least 3:1 (height to diameter at base), at least about 2:1, or atleast about 1:1. A particularly preferred shape for the microprojectionsis a cone with a polygonal bottom, for example hexagonal orrhombus-shaped. Other possible microprojection shapes are shown, forexample, in U.S. Published Patent App. 2004/0087992. Microprojectionsmay in some cases have a shape which becomes thicker towards the base,for example microprojections which have roughly the appearance of afunnel, or more generally where the diameter of the microprojectiongrows faster than linearly with distance to the microprojection's distalend.

The number of microprotrusions in the array can be at least about 50, atleast about 100, at least about 500, at least about 1000, at least about1400, at least about 1600, or at least about 2000. The area density ofmicroprotrusions, given their small size, may not be particularly high,but for example the number of microprotrusions per cm² may be at leastabout 50, at least about 250, at least about 500, at least about 750, atleast about 1000, or at least about 1500.

The array of microprotrusions can formed by providing a mold withcavities corresponding to the negative of the microprotrusions,compression molding the mixture of the melted degradable polymermaterial and viral nanoparticles, demolding the resulting array from themold.

In some embodiments, it may be desired that the microprojections of thearray detach from the array following insertion of the array into skin.Detachable microprojections may be accomplished by a number ofapproaches. A layered approach, for example, may be used in which thearray is composed of multiple layers, and a layer comprising theattachment areas of the microprojections to the array is more readilydegradable than other layers. For example, the layer comprising theattachment areas of microprojections to array may be one which is morerapidly hydrated than the other layers.

Alternatively, an array made of a homogeneous material may be employed,in which the material is more readily degradable at lower pH's. Arraysmade of such a material will tend to degrade more readily near theattachment points because these, being closer to the surface of theskin, are at a lower pH than the distal ends of the microprojections.(The pH of the skin's surface is generally lower than that of the skinfurther inwards, pH being for example approximately 4.5 on the surfaceand approximately 6.5 to 7.5 inward.)

Materials whose solubility is dependent on pH can be, for example,insoluble in pure water but dissolve in acidic or basic pH environment.Using such materials or combination of materials the arrays can be madeto differentially biodegrade at skin surface (pH approximately 4.5) orinside skin. In the former, the whole array can biodegrade while inlatter the microneedle portion of the array will biodegrade whilesubstrate can be removed away.

Microneedle arrays made of materials with pH dependent solubility mayhave additional advantages besides facilitating detachment anddifferential absorption. For example, they may simplify packaging andhandling because of their moisture resistance and rapid hydration andbioadhesion in the buffered acidic or basic environment of the skin.

Microprojection arrays may also be made in which the microprojectionshave a biodegradability which varies with temperature over the range ofexpected use conditions, for example in the range of about 25° C. toabout 40° C. This may be achieved, for example, by the use ofthermosensitive or thermoresponsive polymers. For example, PLGAbiodegrades more slowly at higher temperatures. Certain Pluronicpolymers are able to solidify with rising temperature. A use for thevariation of degradability with temperature is, for example, due to thefact that the microprojections when inserted in skin will tend to havetheir distal ends at a higher temperature than the portions closer tothe base, including the portions (if any) which are not inserted intoskin and are thus at a temperature closer to the ambient temperature.The use of a temperature-dependent biodegradability thus offers afurther way to tailor the biodegradability along the length of themicroprojections.

In another embodiment, as described in the examples, the nanoparticleconstruct can be in the form of a plurality of microparticles that canbe formed, for example, from cry-milled extruded substrate that includesthe virus or virus like particles encapsulated in the degradable polymermatrix. The size of the microparticles can be, for example, about 1 μm,about 10 μm, about 25 μm, about 50 μm, about 100 μm or more. In someembodiments, the size of the particles can be about 1 μm to about 100μm, or about 10 μm to about 25 μm.

In some embodiments, the nanoparticle construct can used to providesustained and/or controlled delivery of virus or virus-like particles toa target tissue in a subject. The nanoparticle construct can be in situdelivered and/or administered to the tissue of the subject. Upondelivery and/or administration of the nanoparticle construct to tissue,the nanoparticle construct can degrade and/or erode by, for example,hydrolysis to release the virus or virus-like particles to the tissue.In particular, virus or virus like particles have been shown topreferentially accumulate in diseased tissue, such as cancer tissue orinflamed tissue (e.g., atherosclerotic blood vessels). While notintending to be bound by theory, it appears that viral nanoparticles canbe taken up by blood components such as macrophage cells of the immunesystem, which subsequently accumulate in diseased tissue (e.g., a tumoror atherosclerotic blood vessel), thereby delivering the virus particleto cells at the disease site.

In some embodiments, administering the nanoparticle construct to asubject can generate an immune response. An “immune response” refers tothe concerted action of lymphocytes, antigen presenting cells,phagocytic cells, granulocytes, and soluble macromolecules produced bythe above cells or the liver (including antibodies, cytokines, andcomplement) that results in selective damage to, destruction of, orelimination from the human body of cancerous cells, metastatic tumorcells, invading pathogens, cells or tissues infected with pathogens, or,in cases of autoimmunity or pathological inflammation, normal humancells or tissues. Components of an immune response can be detected invitro by various methods that are well known to those of ordinary skillin the art.

Other embodiments described herein relate to methods of treating cancerin a subject in need thereof by administering in situ to cancer of thesubject a therapeutically effective amount of the nanoparticleconstruct, which includes a plurality of virus or virus-like particlesencapsulated within a melt processable biodegradable polymer matrix.While not intending to be bound by theory, it appears that the virusparticles or virus like particles, such as plant virus or virus-likepartiles, have an anticancer effect as a result of eliciting an immuneresponse to the cancer. “Cancer” or “malignancy” are used as synonymousterms and refer to any of a number of diseases that are characterized byuncontrolled, abnormal proliferation of cells, the ability of affectedcells to spread locally or through the bloodstream and lymphatic systemto other parts of the body (i.e., metastasize) as well as any of anumber of characteristic structural and/or molecular features. A “cancercell” refers to a cell undergoing early, intermediate or advanced stagesof multi-step neoplastic progression. The features of early,intermediate and advanced stages of neoplastic progression have beendescribed using microscopy. Cancer cells at each of the three stages ofneoplastic progression generally have abnormal karyotypes, includingtranslocations, inversion, deletions, isochromosomes, monosomies, andextra chromosomes. Cancer cells include “hyperplastic cells,” that is,cells in the early stages of malignant progression, “dysplastic cells,”that is, cells in the intermediate stages of neoplastic progression, and“neoplastic cells,” that is, cells in the advanced stages of neoplasticprogression. Examples of cancers are sarcoma, breast, lung, brain, bone,liver, kidney, colon, and prostate cancer. In some embodiments, thenanoparticle constructs are used to treat cancer selected from the groupconsisting of but not limited to melanoma, breast cancer, colon cancer,lung cancer, and ovarian cancer. In some embodiments, the virusparticles are used to treat lung cancer.

In some embodiments, the in situ administration of the nanoparticleconstruct can be proximal to a tumor in the subject or directly to thetumor site to provide a high local concentration and sustained and/orcontrolled release of the virus or virus like particles in the tumormicroenvironment. The method represents a type of in situ vaccination,in which application of an immunostimulatory reagent directly to thetumor modifies the tumor microenvironment so that the immune system isable to respond to the tumor.

In some embodiments, the method can further include the step of ablatingthe cancer. Ablating the cancer can be accomplished using a methodselected from the group consisting of cryoablation, thermal ablation,radiotherapy, chemotherapy, radiofrequency ablation, electroporation,alcohol ablation, high intensity focused ultrasound, photodynamictherapy, administration of monoclonal antibodies, immunotherapy, andadministration of immunotoxins.

In some embodiments, the step ablating the cancer includes administeringa therapeutically effective amount of an anticancer agent to thesubject. Examples of anticancer agents include angiogenesis inhibitorssuch as angiostatin K1-3, DL-α-difluoromethyl-ornithine, endostatin,fumagillin, genistein, minocycline, staurosporine, and (±)-thalidomide;DNA intercalating or cross-linking agents such as bleomycin,carboplatin, carmustine, chlorambucil, cyclophosphamide, cisplatin,melphalan, mitoxantrone, and oxaliplatin; DNA synthesis inhibitors suchas methotrexate, 3-Amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin,cytosine P-D-arabinofuranoside, 5-Fluoro-5′-deoxyuridine,5-Fluorouracil, gaciclovir, hydroxyurea, and mitomycin C; DNA-RNAtranscription regulators such as actinomycin D, daunorubicin,doxorubicin, homoharringtonine, and idarubicin; enzyme inhibitors suchas S(+)-camptothecin, curcumin, (−)-deguelin, 5,6-dichlorobenz-imidazole1-β-D-ribofuranoside, etoposine, formestane, fostriecin, hispidin,cyclocreatine, mevinolin, trichostatin A, tyrophostin AG 34, andtyrophostin AG 879, Gene Regulating agents such as5-aza-2′-deoxycitidine, 5-azacytidine, cholecalciferol,4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, alltrans-retinal, all trans retinoic acid, 9-cis-retinoic acid, retinol,tamoxifen, and troglitazone; Microtubule Inhibitors such as colchicine,dolostatin 15, nocodazole, paclitaxel, podophyllotoxin, rhizoxin,vinblastine, vincristine, vindesine, and vinorelbine; and various otherantitumor agents such as 17-(allylamino)-17-demethoxygeldanamycin,4-Amino-1,8-naphthalimide, apigenin, brefeldin A, cimetidine,dichloromethylene-diphosphonic acid, leuprolide,luteinizing-hormone-releasing hormone, pifithrin, rapamycin,thapsigargin, and bikunin, and derivatives (as defined for imagingagents) thereof.

In some embodiments, the step ablating the cancer includes immunotherapyof the cancer. Cancer immunotherapy is based on therapeuticinterventions that aim to utilize the immune system to combat malignantdiseases. It can be divided into unspecific approaches and specificapproaches. Unspecific cancer immunotherapy aims at activating parts ofthe immune system generally, such as treatment with specific cytokinesknown to be effective in cancer immunotherapy (e.g., IL-2, interferon's,cytokine inducers).

When used in vivo, the nanoparticle constructs can be administered as apharmaceutical composition, and a pharmaceutically acceptable carrier.The nanoparticle constructs, or pharmaceutical compositions comprisingthese constructs, may be administered by any method designed to providethe desired effect. Administration may occur enterally or parenterally;for example, orally, rectally, intracisternally, intravaginally,intraperitoneally or locally. Parenteral administration methods includeintravascular administration (e.g., intravenous bolus injection,intravenous infusion, intra-arterial bolus injection, intra-arterialinfusion and catheter instillation into the vasculature), peri- andintra-target tissue injection, subcutaneous injection or depositionincluding subcutaneous infusion (such as by osmotic pumps),intramuscular injection, intraperitoneal injection, intracranial andintrathecal administration for CNS tumors, and direct application to thetarget area, for example by a catheter or other placement device.

One skilled in the art can readily determine an effective amount of thenanoparticle constructs to be administered to a given subject, by takinginto account factors such as the size and weight of the subject; theextent of disease penetration; the age, health and sex of the subject;the route of administration; and whether the administration is local orsystemic. Those skilled in the art may derive appropriate dosages andschedules of administration to suit the specific circumstances and needsof the subject. For example, suitable doses of the virus particles to beadministered can be estimated from the volume of cancer cells to bekilled or volume of tumor to which the virus particles are beingadministered.

Useful dosages of the nanoparticle constructs can be determined bycomparing their in vitro activity and the in vivo activity in animalmodels. Methods for extrapolation of effective dosages in mice, andother animals, to humans are known in the art. An amount adequate toaccomplish therapeutic or prophylactic treatment is defined as atherapeutically- or prophylactically-effective dose. In bothprophylactic and therapeutic regimes, agents are usually administered inseveral dosages until an effect has been achieved. Effective doses ofthe nanoparticle constructs can vary depending upon many differentfactors, including means of administration, target site, physiologicalstate of the patient, whether the patient is human or an animal, othermedications administered, and whether treatment is prophylactic ortherapeutic.

Advantageously, the nanoparticle construct can provide a slow-releaseand/sustained formulation of the virus or virus like particles as an insitu vaccine that maintains sustained immune stimulation without theneed for repeat injections. The release of the plant virus or virus likeparticles can be constant and sustained for about 1 day, 2 days, 3 days,4 days, 5 days, 6 days, 1 week, 2 weeks or more. The constant releasecan be sustained between subsequent nanoparticle administrations.Maintaining a constant immunostimulatory effect can reduce the number ofadministrations, enhancing their effectiveness. The release of the virusor virus-like particles having from the degradable polymer matrix can beat least partially defined by the swelling and degradation rate of thedegradable polymer material under physiological conditions.

In some embodiments, a dose of the virus or virus-like particles can becoadministered with the nanoparticle construct in situ to cancer of thesubject to provide an initial immune response prior to sustained releaseof the virus or virus-like particles from the nanoparticle construct.These combined strategies can maintain the single administration vaccinenature of the administration of the nanoparticle construct and willlikely improve treatment of the cancer.

In still other embodiments, the nanoparticle construct can includedegradable polymer matrix and a plurality rod-shaped viral nanoparticlesthat are encapsulated in the matrix. The rod-shaped viral nanoparticlescan be used as carriers to deliver at least one agrochemical agent oringredient in a controlled and targeted manner for agriculturalapplications. Rod-shaped plant viral nanoparticles can provide aneconomically and environmentally viable alternative to conventionalsynthetic nanoparticles. Plant viral nanoparticles can be produced inlarge quantities in a short time for a relatively low price. Inaddition, plant viral nanoparticles are exceptionally robust to theharsh environment of crop fields, biodegradable, as well asbiocompatible and noninfectious, making them safe to use on industrialcrops.

In some embodiments, a melt processed nanoparticle construct can includea plurality of rod-shaped viral nanoparticles encapsulated in adegradable polymer matrix and at least one agrochemical agent that isconjugated to and/or loaded on and/or within the viral nanoparticles.The rod shaped viral nanoparticle can have an exterior surface and aninterior surface that extend from a first end to a second of therod-shaped viral nanoparticle. The interior surface can define a channelthat extends through rod-shaped viral nanoparticle from the first end tothe second end. The channel can include the viral genome or lack theviral genome. The agrochemical agent can be conjugated to an interiorand/or exterior surface of the viral nanoparticle.

In some embodiments, the viral nanoparticles include Virgaviridae virusparticles. In other embodiments, the viral nanoparticles include one atleast one viral nanoparticle of the Tobamovirus species. Particularexamples include, but are not limited to, tobacco mild green mosaicvirus nanoparticles and tobacco mosaic virus nanoparticles.

In other embodiments, the agrochemical agent can be covalently ornoncovalently coupled and/or conjugated to the viral nanoparticles orloaded on or within the degradable polymer matrix of the nanoparticleconstruct. In one example, positively charged agrochemical agents can benon-covalently loaded onto negatively charged interior or exteriorsurfaces of the rod-shaped viral nanoparticles by electrostaticinteractions between the positively charged agrochemical and carboxylategroups of exposed aspartic acid and glutamic acid residues on theinterior and exterior surface of the rod-shaped viral nanoparticlesprior to melt processing the rod-shaped viral nanoparticles anddegradable polymer material. In another example, agrochemical agents canbe covalently bound to chemically modified carboxylate groups of exposedglutamic acid, aspartic acid, and tyrosine residues on the interior orexterior surface of the rod-shaped viral nanoparticles.

The agrochemical agent conjugated to the interior and/or exteriorsurface of the rod-shaped viral nanoparticle can be selected from thegroup consisting of nematicides, fungicides, herbicides, pesticides,acaricides, rodenticides, plant growth regulators, nutrients, pestrepellents, and combinations thereof.

In some embodiments, the nanoparticle constructs can be formulated as aplurality of particles to facilitate delivery of the nanoparticleconstruct to a pest, plant, plant organ, plant propagation material, ora surrounding area thereof.

Other embodiments described herein relate to a method of treating aplant. The method can include applying a nanoparticle construct asdescribed herein to the plant in a treatment effective amount. Suchplants are generally angiosperms or gymnosperms, and in some embodimentsare monocots or dicots. In some embodiments, the plant is wheat, corn(maize), soybean, cotton, cassava, potato, sweet potato, bananas,citrus, strawberries, tomato, coffee, carrots, peppers, turf grass, orgreenhouse ornamentals, taro, oats, barley, cereal rye, breadfruit, pea,rice, yams, garbanzo (chickpea), Jerusalem artichoke, or lentil.

In some embodiments, the plant may be in the form of a plant part, suchas leaves, flowers, stems, roots, tubers, fruits, and seeds.

In other embodiments, the composition is applied in an amount effectiveto combat nematode parasitism on said plant.

In some embodiments, the nanoparticle constructs including therod-shaped viral nanoparticles loaded with the agrochemical agent canhave greater soil mobility than the agrochemical agent alone. This canprovide agrochemical agent loaded rod-shaped VNPs with enhancedpenetration through soil to reach pests, such as nematodes, that feed onthe roots of plants.

Examples have been included to more clearly describe particularembodiments of the invention. However, there are a wide variety of otherembodiments within the scope of the present invention, which should notbe limited to the particular examples provided herein.

EXAMPLE 1

In this Example, we used melt encapsulation to create solid-statepoly(lactic-co-glycolic acid) (PLGA) implants laden with Qβ forapplications in slow-release vaccine development. Processing parametersrelevant to extrusion or injection molding could be tuned to maintainparticle integrity post-processing, providing a window for scale up tocommercial polymer processing equipment. Of utmost importance, singledose implants perform equivalently to traditional vaccine administrationschedules.

Material and Methods Preparation of PLGA/Protein Implants

Poly(lactic-co-glycolic acid) (PLGA), 8 kDa polyethylene glycol (8KPEG),and 20 kDa PEG (20KPEG) were individually ground manually with a mortarand pestle twice, 10 minutes each time, into a fine powder. The PLGApowder consisted of particles with an average length of 185.8 ±89.1 μmas determined via SEM image analysis. PLGA was mixed with theappropriate weight percent of lyophilized Qβ and PEG (if added) viarepeated vortexing in a 2 mL Eppendorf tube. Formulations were asfollows with all percentages expressed as a weight percent: PLGA/1%Q(3;PLGA/1%Qβ/10%8KPEG; PLGA/1%Qβ/10%20KPEG, PLGA/5%Qβ, PLGA/10%Qβ. Twodifferent custom built aluminum syringe-die were used for meltprocessing of the blends to minimize material input. Both syringe-diesystems consisted of a cylinder with a circular 1 mm exit diameter thatwas wrapped with heating tape, combined with a digital control elementto provide constant heating. The die used for melt encapsulation ofsamples for in vitro testing utilized polypropylene BD™ LUER LOK™syringes which were filled with 500-200 mg of the PLGA/Qβ blends andheated at 95 ° C. as determined by a glass thermometer (99.9° C. averagealong the temperature profile as determined via an infrared thermometer)for 10 minutes. The melted PLGA/Qβ blend was flowed through the dieusing a syringe pump with a velocity of 3 mm s⁻¹ (-2.35 mm³ s⁻¹volumetric flow rate). The resulting cylindrical implants had diametersranging from 1.0-1.3 mm. Melt encapsulation of ClearColi® produced Qβfor in vivo testing was performed with a cylinder manufactured to fitpolypropylene 1 mL volume Norm-Ject syringes. The die still consisted ofa circular 1 mm hole. This barrel was used to minimize materials due tothe lower yield of ClearColi® produced particles. The syringe was filledwith 50-100 mg of the appropriate PLGA/Qβ blend and extruded in the samemethod as previously described. There was no difference observed inimplant diameter or particle integrity between samples fabricated withdifferent barrels.

Shear Application

Shear application was performed by loading 150-300 mg of PLGA/1%Qβ ontoa 25 mm wide parallel plate rheometer at 95° C. Samples were allowed toequilibrate for 5 minutes, then the top plate was lowered to a gap of0.45 mm and shear rates from 0.1-50 s⁻¹ were applied for 3 minutes. Thesample was recovered from the rheometer post-shear and the Qβ wasrecovered and analyzed via the extraction method previously described.The viscosity of the samples was also measured during this process andfound to be in the range of 120-130 Pa·s, with an average of 128 Pa·s.

Radius Shear Dependency and Peclet Number Calculations

Qβ samples recovered post-shear application were analyzed via DLS andFPLC. Weight average hydrodynamic radii were calculated from the DLSdata for samples subjected to 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, and 50s⁻¹. Samples subjected to 25 and 50 ⁻¹ exhibited extensive particlebreakup when analyzed via FPLC. The breakup product was assumed to becoat protein dimers, which exhibit a radius of 3.21 nm estimated fromthe crystal structure (PDB: 1QBE). This estimate is similar to thehydrodynamic radius of green fluorescent protein (2.8 nm), which is ofsimilar molecular weight to the coat protein dimer (27 and 28 kDarespectively). The ratio of intact particles to coat protein dimers wascalculated via curve fitting of the two major curves observed in theFPLC. The ratio of intact particles was multiplied by the weight averageradius determined via DLS and added to the ratio of coat protein dimermultiplied by 3.21 nm to give an average radius of species in the 25 and50 s⁻¹ samples, as shown by the equation below.

R _(Ave)-32 (R _(Ave.DLS))*(%_(Particle))+(3.21 nm)*(%_(Dimer))

Where: R_(ave)=average radius for samples subjected to 25 and 50 s⁻¹shear rates

R_(Ave, DLS)=mass average radius calculated from the DLS result

%_(Particle)=percentage of particle calculated from curve fitting of theFPLC

%_(Dimer)=percentage of dimer calculated from curve fitting of the FPLC

The weight average radius was divided by the weight average radius of Qβthat had been extracted from PLGA/1%Qβ samples that had not beensubjected to shear. This result was plotted as the radius of shearapplied samples to the initial radius versus shear rate.

This result was non-dimensionalized by calculating the Peclet number foreach shear rate. The Peclet number (Pe) is a dimensionless number of theratio of convective forces vs the diffusive forces in a fluid system.The Peclet number was calculated as the ratio of shear stress applied onthe particles over the diffusive forces estimated by the Stokes-Einsteinequation, as shown by the equation below.

${Pe} = \frac{6{\pi\eta}\overset{.}{\gamma}R^{3}}{k_{b}T}$

Where: η=viscosity of the polymer melt (Pa·s)

{dot over (γ)}=shear rate applied to the system (s⁻¹)

R=weight average radius of the particles before shear application (m)

k_(b)=Boltzmann's constant (J·K⁻¹)

T=temperature of the system (K)

The resulting plot of particle radius of shear applied samples to theinitial radius versus Peclet number is useful in relating theaggregation behavior of Qβ during melt encapsulation to other polymersystems with different viscosities and processing temperatures.

Shear Application Thermal Analysis Calculations

Mathematical analysis was performed to estimate the total applied energyto the system during shear application and correlate it to the observedparticle breakup. Qβ particle breakup into free dimers involves thebreakage of disulfide bonds between adjoining dimers on the particle,with each dimer containing 4 disulfide linkages and one particlecontaining 90 coat protein dimers. The bond dissociation energy of adisulfide bond is typically 251 kJ/mol. Thus, the theoretical energy ofall disulfides per particle was calculated to be 45,180 kJ/mole ofparticle. Integration of the first endothermic peak on the DSCthermogram (from 84 to 172° C.) as shown by the equation below, which isspeculated to be disulfide breakup, yielded a value of 43,860 kJ/molparticle in good agreement with the theoretical value.

$E_{disulfide} = {( {MW}_{Q\;\beta} )( \frac{\Delta\; T}{s} )^{- 1}{\int_{T_{1}}^{T_{2}}{( \frac{W}{g} ){dT}}}}$

Where: E_(disulfide)=total enthalpy of disulfides per mole of particle(J/mol Qβ)

MW_(Qβ)=molecular weight of Qβ=2,556,000 g/mol

ΔT/s =heating rate of the DSC study, 0.333 K/s

mW/g =heat flux of the DSC sample per gram (W/g)

The value determined via DSC integration was used to calculate the totaldisulfide bond energy present based on the mass of Qβ present in eachshear application sample. The moles of PLGA in the system was calculatedbased on the mass of PLGA in the system and an average molecular weightof 12.5 kDa.

The total applied energy to the system during shear application wascalculated as the sum of the energy applied by shear stress and thermalenergy with the effects of shear heating taken into account utilizingthe equations shown below. The energy values were normalized by thetotal disulfide bond energy present in each sample.

E_(shear)=η{dot over (γ)}V_(system)

E _(thermal) =k _(b) N _(A)(T _(applied) +ΔT_(shear))(mol_(Qβ)+mol_(PLGA))

Where: η=viscosity of the polymer melt (Pa.·s)

{dot over (γ)}=shear rate applied to the system (s⁻¹)

V_(system)=total volume of Qβ and PLGA (m³)

k_(b)=Boltzmann's constant (J·K⁻¹)

N_(A)=Avagadro's number

T_(applied)=temperature during shear application (K)

ΔT_(shear)=temperature increase due to shear heating (K)

mol_(Qβ)=moles of Qβ in the system

mol_(PLGA)=moles of PLGA in the system

Immunization and ELISA Analysis

Prior to immunization studies, 3 male Balb/c mice aged 7 weeks wereimplanted subcutaneously with ˜0.5 cm of neat PLGA cylinder via puncturewith a 16 gauge needle and insertion with forceps. The mice weremonitored for 4 weeks and exhibited swelling at the site of insertionfor 2 weeks after insertion, which subsequently subsided. The mice didnot exhibit any adverse health or behavioral response to theimplantation of the neat PLGA cylinders. For standard immunization, maleBalb/c mice (Charles River) aged 7 weeks (n=5) were immunized 3 times ondays 0, 14, and 28 with 50 μg Qβ in 100 μL sterile PBS throughsubcutaneous injections behind the neck using a 29G insulin syringe. TheQβ was produced in ClearColi E. coli cells that contain a modifiedlipopolysaccharide (LPS) outer membrane that does not elicit an immuneresponse in mice. Blood (˜100 82 L) was drawn prior to the firstimmunization and on a weekly to biweekly basis via the retro-orbitalplexus using heparinized capillary tubes and collected in GreinerBio-One VACUETTE™ MiniCollect™ tubes. Serum was separated bycentrifuging blood samples at 14,800 rpm, 4° C., for 10 min and storedat 4° C. until analyzed via enzyme-linked immunosorbent-assay (ELISA).For implant immunization, male Balb/c mice (Charles River) aged 7 weeks(n=5) had 0.5 cm (˜8 mg) of PLGA/10%Qβ inserted into the subcutaneousspace on the neck via puncture with the tip of a 16-gauge needle andinsertion with forceps. The amount of implanted material was chosen todeliver roughly the same amount of Qβ over the first 28 days as the miceimmunized via subcutaneous injection based on the in vitro releaseprofile, with ˜0.8 mg of implant correlating to ˜150 (ig of released Qβover 30 days. Orbital bleeds were conducted as previously described onthe same days as the standard immunization schedule mice. All mice wereboosted at day 65 with 50 μg of Qβ. After day 75, all mice wereeuthanized, and the subcutaneous space was examined. No implant materialwas present in any of the implanted mice and no extensive scar tissuewas present compared to non-implanted mice.

The anti-Qβ IgG response was measured by first coating Nunc Maxisorp96-well plates with 2 μg of Qβ in 200 (IL of sterile PBS, pH 7.4 at 4°C. overnight. The wells were then blocked with 200 (IL of blockingbuffer (2.5% w/v dry milk, 25% neonatal calf serum in PBS, pH 7.4) at37° C. for 1 hour. The wells were then incubated with mouse sera atdilutions from 1:100 to 1:1000000 in 100 (IL blocking buffer for 2 hoursat 37° C. The wells were then incubated with 100 (IL of a 1:1000dilution in blocking buffer of alkaline-phosphatase labeled goatanti-mouse IgG for 1 hour at 37° C. The wells were washed between eachincubation step using 3×250 (IL of 0.1% w/v Tween-20 in PBS, pH 7.4. Thewells were developed using 100 μL of 1-step PNPP substrate at 4° C. for10 minutes. The reaction was stopped with 100 μL of 2 M NaOH and theabsorbance was read at 405 nm in triplicate for each sample. Theend-point titer value was determined by comparison to a statisticallydefined cutoff value based on the pre-bleed measurements of 10 mice anda confidence level of 99%. Values are expressed as the average andstandard deviation of 5 mice.

Murine anti-Qβ IgG subtypes were determined via the ELISA methoddescribed above with alkaline-phosphatase labeled goat anti-mouse IgG1,IgG2a, and IgG2b used for detection. Percentages are expressed as theaverage and standard deviation of 5 mice.

Poly(lactic-co-glycolic acid) (EXPANSORB® 10P019, 50:50 PLGA, inherentviscosity 0.15-0.25 dlg⁻¹, 5-20 kDa) was purchased from PCAS. Potassiumphosphate monobasic anyhdrous, potassium phosphate dibasic anydrous,sodium phosphate dibasic hetptahydrate, Gibco 1× PBS pH 7.4, butanol,Miller LB Broth, D-sucrose, guanidine hydrochloride, sodium dodecylsulfate, and isopropyl (3-D-1-thiogalactopyranoside, kanamycin,spectinomycin, sodium azide, ethyl acetate, neonatal calf serum, 1-stepPNPP substrate, Tween-20, albumin standard, and sodium hydroxide werepurchased from Fisher Scientific. Poly(ethylene glycol) (M.=20000) waspurchased from Alfa Aesar. Poly(ethylene glycol) (M_(n) =8000) waspurchased from Amresco. Bradford reagent was purchased from VWR. Goatanti-mouse IgG-alkaline phosphatase antibody was purchased from LifeTechnologies. Goat anti-mouse IgG2a, IgG2b and IgG1-alkaline phosphataseantibodies were purchased from Novus Biologics. All reagents were useddirectly, without further purification.

Results

Qβ VLPs were expressed recombinantly in E. coli and purified withtypical yields of ˜50-100 mg per liter of culture. Chromatographicanalysis verified the purity as a single peak in the size-exclusionchromatogram with no aggregates, free protein, or free RNA present (FIG.2A). The purified VLPs exhibited a hydrodynamic radius of ˜15 nmdetermined by dynamic light scattering (DLS) and verified bytransmission electron microscopy (TEM). TEM analysis yielded an averageradius of 13.2 nm determined via image analysis (FIG. 2B, 2C). Thesmaller radius observed in the TEM micrographs is a result ofdehydration during TEM preparation versus the hydrodynamic radiusmeasured by DLS. The VLPs were dialyzed into deionized water andlyophilized to yield a fluffy white powder. Resuspension of the powderinto PBS and analysis by DLS and fast protein liquid chromatography(FPLC) indicated that lyophilization yielded no negative effect on Qβand did not result in the formation of aggregates or disassembly of theparticles.

Dynamic scanning calorimetry (DSC) of Qβ was conducted to determinethermal transitions that may occur in the processing window for PLGA.The DSC thermogram of the freeze-dried Qβ yielded two endothermic peaks,at 130 and 236° C. respectively. The peak at 130° C. was attributed tothe break-up of the disulfide bonds that stabilize coat protein dimersin the icosahedral Qβ structure. Disulfide bond dissociation has beenobserved in the range of 80-160° C. in solid-state rubber vulcanizationand self-healing materials. The endothermic peak at 236° C. can beattributed to the dissociation of coat protein dimers and denaturationof the coat proteins. Previous DSC studies of lysozyme, glycinin, andhuman growth hormone in the solid-state have indicated proteindenaturation over temperature ranges of 180-200° C. The higherdenaturation temperature observed with lyophilized Qβ was likely due tostrong intermolecular attractions associated with dimer stability. TheDSC study of Qβ provided insight into the denaturation process duringheating in the solid state and ensured that no major denaturationprocesses occurred in the processing window of 80-100° C. typically usedfor melt-encapsulation of proteins with PLGA.

Qβ laden implantable polymeric materials were manufactured viamelt-encapsulation with PLGA utilizing a lab-built syringe-die extrusiondevice (FIG. 1). The syringe-die extruder allows for melt-processing ofmaterial amounts as low as 100 mg. This provides access tolaboratory-scale experiments since commercial small scale extrudersrequire a minimum of 5 grams for effective processing. The low amount ofmaterial required makes this a valuable tool for pilot-scale experimentsusing high-value materials for initial testing. PLGA and Qβ powders werecombined and vortexed repeatedly to homogenize the mixture. The mixturewas then loaded into a syringe, placed in the extrusion device at 95° C.for 10 minutes, and then pushed through a 1 mm cylindrical die to yieldcylindrical opaque materials. The DSC thermogram of PLGA indicated aglass transition temperature of 37° C. with a small melting endotherm at41° C. The processing temperature was the lowest that gave the mosthomogenous cylindrical shape, with no observable aggregated portions ofQβ powders along the length of the extruded material. PLGA materialsloaded with 1, 5, and 10 w/w% Qβ were prepared via this method.

Following implant fabrication, Qβ was extracted to determine particleintegrity, aggregation state, and capsid disassembly. Extraction of Qβprocessed at 1 wt % was performed utilizing ethyl acetate to dissolvethe polymer and the remaining Qβ was resuspended in PBS. Treatment oflyophilized Qβ with ethyl acetate and resuspension yielded no aggregatedor disassociated species. Qβ extracted after melt-encapsulation wasrecovered at ˜90% and analyzed via DLS and FPLC (FIG. 2D and 2E). DLSand FPLC both indicated the formation of small amounts of aggregatedspecies after melt-encapsulation. The aggregated species apparent in theDLS histogram span from -30 to 70 nm in radius, indicating small-scaleaggregated clusters of 2 to 5 VLPs. The DLS data was plotted as a masspercentage to better represent the proportion of aggregated species aspercent intensity skews toward larger species. The aggregates made up˜16% of the recovered VLPs calculated from the mass percentagedistribution. The peak at 10 mL retention volume in the FPLCchromatogram appeared at the void volume of the column with allaggregated species eluting at this volume. Curve fitting and integrationof the FPLC curves yielded a relative percentage of 12% aggregated VLPs.Minor peak broadening was observed in the portion of the curvecorresponding to dispersed Qβ. Both DLS and FPLC analysis yieldedsimilar values of aggregated species and indicate roughly 85% of VLPsremain dispersed and intact after melt-encapsulation with PLGA at 95° C.TEM further verified that the particles retained their characteristicicosahedral shape after melt-encapsulation and the radius determined viaimage analysis was 13.3 nm, in good agreement with the size determinedvia TEM prior to melt-encapsulation (FIG. 1F).

The dispersion of Qβ within the PLGA matrix in the solid state wasexplored through elemental mapping using energy dispersive X-rayspectroscopy coupled with scanning electron microscopy (EDS-SEM).Aggregation and segregation of proteins within the polymer matrix canoccur during melt-processing, resulting in regions of protein rich andprotein poor areas through the cross-section of the material. This poordispersion of protein can result in irreversible aggregation anddenaturation, as well as an inconsistent release profile due to burstrelease when the aggregated regions are exposed to solvent during matrixdegradation. All of these factors would negatively affect the desiredproperties of the Qβ/PLGA materials, thus the effect of loading level onthe dispersion of Qβ within PLGA in the solid state was studied toensure the particles were properly dispersed throughout the matrix.EDS-SEM was a valuable tool for studying dispersion, as it allowed forthe visualization of distinct elemental signals through a cross-sectionof material. SEM images of the freeze-fractured cylindrical materialswere obtained and the distribution of the EDS sulfur K-series signalthroughout a 12.5×12.5 μm cross-section was obtained (FIG. 3). Thesulfur K-series signal was chosen for EDS analysis since it is unique tothe protein component in the implant. Qualitative assessment of mappingfrom neat PLGA indicated very little background signal. The mapping ofPLGA loaded with 1, 5, and 10% Qβ resulted in good qualitativedispersion and no segregation of sulfur poor and rich regions. Thenumber of spots corresponding to sulfur signal does not increaseproportionately to the loading level, however the color intensity of thespot is dependent on the amount of sulfur in the system. Signalthresholding of the images to 50% of maximum signal yielded images thatmore clearly show the increase in sulfur signal in response to increasedloading levels. Furthermore, the quantitative sulfur signal from the EDSspectrum was 0.08, 0.33, and 0.82 wt % for loading levels of 1, 5, and10 wt %, respectively, indicating that sulfur content cariesproportionally with loading level. Quantitative dispersion analysis wasperformed utilizing ImageJ to determine the cumulative distributionfunction (CDF) of particles based on the nearest neighbor andpoint-to-point distance analysis. The CDF gives the probability that aparticle-particle distance is equal to or less than the distance on theplot. The nearest neighbor and point-to-point distance cumulativedistributions followed similar profiles for all loading levels. Thenearest neighbor analysis gave relatively small values for nearestneighbor distance, with 75% of the particles having a nearest-neighborless than 0.5 μm away. The linearity of the point-to-point distance plotis also indicative of the particles being well dispersed, as thedistance of the points away from each other would increase sporadicallyif they were aggregated into clusters. Overall, loading levels of 1, 5,and 10 wt % Qβ did not form aggregated regions within the PLGA duringmelt-encapsulation based on the qualitative and quantitative results.This result indicated that Qβ can be melt-encapsulated at 1, 5, and 10wt % with PLGA without aggregation within the matrix, ensuring that therelease of Qβ from the matrix will not be adversely effected and will bereplicable.

Qβ VLP laden cylindrical polymeric devices were successfullymanufactured via melt-encapsulation and the VLPs maintained particleintegrity following extraction. However, most commercial manufacturingprocesses for polymer nanocomposites typically follow a two-stepprocess; a masterbatch of the composite is compounded followed bygeometric molding. The syringe extrusion process described above isrepresentative of the initial compounding step in the manufacturing ofpolymeric nanocomposites. Next, we sought to evaluate how VLP integrityin the masterbatch could be maintained in downstream processes such asextrusion, injection molding, or compression molding. All of theseprocesses introduce the composite to similar stress forces, namelypressure and shear forces. Thus, the cylindrical Qβ/PLGA material wassubjected to a melt-press, to simulate pressure, or to shear applicationusing a rheometer. The 1 wt % loaded samples were utilized forpost-processing studies as they could be produced in the highest amountdue to the lower amount of Qβ needed for material processing.

Typical compression molding is performed by pumping material into acavity at high pressures for a set amount of time to mold the materialinto the desired shape. The pressures and times range depending on thepolymer, mold, and desired device properties, but generally a pressurerange of 500-2000 psi and cycle times of 2-5 minutes are utilized. Amelt-press was used to apply a pressure of 1200 psi for 5 minutes at 95°C., an intermediate range for compression molding. The melt-pressed andextracted VLPs were analyzed via DLS and FPLC to determine the integrityand aggregation state of extracted and recovered Qβ (FIG. 4). Theresulting DLS histogram indicated an increase in both the amount andsize of aggregated particles. Aggregates in the range of 40 to 100 nmradius were observed in the DLS data, corresponding to systems of 3 to 7particle aggregates. The percentage of recovered VLPs that wereaggregated was 25.5%, an increase of -10% from the initialmelt-encapsulation step. The FPLC chromatogram of the recovered Qβindicated the presence of aggregates and intact particles with peakmaxima at 10 mL and 18 mL respectively. The peak centered at 18 mL hadconsiderable broadening towards lower retention volume. A minor tail wasobserved at higher elution volumes indicating some particle breakup,however this was negligible compared to the remainder of the population.Relative integration of curves fit at 10 mL and 18 mL resulted in 13% ofthe recovered particles being aggregated. The discrepancy between thepercentage of aggregated species between the DLS and FPLC is a result ofthe curve in the FPLC not being a true Gaussian curve, thus skewing thecurve fitting result. The simulated compression molding conditionsresulted in a modest increase in aggregated species from initialmelt-encapsulation (˜10%) and the majority of Qβ remained as singledispersed particles, demonstrating this system can be suitable forprocessing via compression molding.

A rheometer was used to apply different shear rates to 1 wt % Qβ loadedPLGA to emulate the shear effects applied during post-processing steps.The range of shear rates chosen were from 0.1-50 s⁻¹, which correlatedto processes with relatively low applied shear. This range is mostcommonly used in compression molding, blow molding, and 3D printingprocesses and relevant in conditions the Qβ/PLGA material would be underduring production of more complex architectures for implantation. Shearwas applied utilizing a rheometer with a parallel plate configurationfor 3 minutes at 95° C. and Qβ was recovered via ethyl acetateextraction. The DLS and FPLC results of Qβ after application of shearrates from 0.1-50 s⁻¹ indicated a three-phase response to increasingshear rates. (FIG. 5). The lowest shear rates from 0.1-1 s⁻¹ resulted inan increase in both the size and amount of aggregates in the recoveredVLPs in response to increasing shear rate. Further increase in appliedshear rate to 2.5-10 s⁻¹ diminished the size and amount of aggregatesobserved. Increasing the applied shear rate to 25 and 50 s⁻¹ yielded noobservable aggregates in the DLS histogram. Analysis of the recovered Qβvia FPLC indicated the same trend observed with DLS and the intensity ofthe aggregate peak at 10 mL increased relative to the Qβ peak at 18 mLafter application of shear rates from 0.1 to 1 s⁻¹.

FPLC chromatograms of samples subjected to shear rates from 2.5 to 10s⁻¹ were also in good agreement with the DLS results, with the aggregatepeak diminishing in intensity as the shear rate increased. All samplessubjected from 0.1 to 10 s⁻¹ exhibited some degree of disassociatedspecies eluting at higher retention volumes. These species likelyconsist of partially disassociated VLPs, free coat protein dimers, andfree RNA. Thus, any application of shear to the samples appears toresult in a degree of disassociation of Qβ, however these are allrelatively minor when compared to the aggregates and single particlesbased on the peak area observed in the FPLC. As shear rates approached10 s⁻¹, a significant reduction in particle aggregates is observed andthe chromatograms show predominately intact particles. Finally, as theshear rate continues to increase to 25 and 50 s⁻¹ extensive capsiddissociation is seen, as evidenced by the predominant peak at 23.2 mL inthe FPLC. Curve-fitting and relative integration of the FPLC curvesyielded values of 35% and 22% of particles maintaining integrity afterapplication 25 and 50 s⁻¹ shear rates respectively. The disassociatedspecies were not observed in the DLS data as the estimated radius of ˜3nm, based on the crystal structure, falls below the limit of detectionfor the instrument. The particle break-up observed at 25 and 50 s⁻¹indicate that care must be taken in applying higher shear rates to theQβ/PLGA material. Processes such as twin-screw extrusion and injectionmolding often have shear rates above100 s⁻¹, which would not be suitablefor this system. However, these limitations could be overcome withslower screw speeds during extrusion to maintain shear rates in theacceptable range to retain particle integrity.

It was evident from the DLS and FPLC analysis that the aggregation stateand integrity of Qβ within PLGA is dependent on the shear rate appliedduring melt-processing. As such, we sought to derive a physical model todetermine particle stability versus aggregation state in varying shearenvironments. The mass average radius of all species in the recoveredVLPs was estimated from the DLS distribution for samples at shear ratesfrom 0.1 to 10 s⁻¹. Shear rates of 25 and 50 s⁻¹ cause extensiveparticle breakup, with the disassociated particles unable to be measuredvia DLS due to the lower limits of detection. Thus, the radius averagefor the 25 and 50 s⁻¹ samples was estimated using the DLS radius forintact particles and the radius of 3.2 nm for coat protein dimer using aglobular estimation of the coat protein dimer from the crystalstructure. These two values were averaged using the percentage of intactparticle and coat protein dimer estimated from the relative integrationof the FPLC curves. The averaged radii were normalized by the averageradius of Qβ recovered before the application of shear (<R>/<R_(o)>) andplotted versus the applied shear (FIG. 6A, top axis). The resulting plotdemonstrated a clear dependence of particle aggregation anddisassociation on applied shear rate. The average particle sizeincreases to 3 times the initial radius with increasing shear rate, witha maximum reached at is⁻¹. Processing particles at shear rates greaterthan 1 s⁻¹ causes a return to the initial radius, until a critical shearrate of 25 s⁻¹ was reached where particle dissociation occurred. Thisinformation is useful for designing post-processing conditions for Qβladen PLGA materials, however the trend in aggregation state observed isonly applicable to this polymer system.

The shear rate relationship was transformed into a Peclet numberrelationship to expand the utility of the data to processing Qβ withother polymer systems and at differing temperatures (FIG. 6A, bottomaxis). The Peclet number is a dimensionless number that represents theratio of convective forces to diffusive forces. The convective forcesare dependent on the shear rates applied and the diffusive forces aredependent on the Brownian motion in the system. This allowed for theestimation of shear forces based on the viscosity of the melted system,the shear rate, and the volume of the system. The Brownian forces wereestimated by the Stokes-Einstein equation, which is directly dependenton temperature. Conversion of the aggregation state relative to thePeclet number generalizes the relationship and allows for the estimationof aggregation state in other shear dependent processes. Understandingthe aggregation state in response to the applied shear, polymerviscosity, and temperature allows for the calculation of relevantprocessing conditions without extensive scouting experiments. Thus, therelationship derived from the shear rate application, aggregationstates, and Peclet number will allow for the determination of processingconditions to create materials with minimal aggregation and particlebreak-up for other polymer systems and temperatures. In this case,Peclet values between ˜5 and 25 resulted in well dispersed singlenanoparticles without dissociation, providing a baseline value fortranslation to alternative systems.

The stability of Qβ during melt-processing is theorized to be due to thehighly interconnected network of disulfide bonds that link coat proteindimers together, forming a thermally and chemically stable covalentlyattached assembly. The extensive particle break-up observed in samplessubjected to 25 and 50 s⁻¹ shear rates was hypothesized to be a resultof the disassociation of disulfide linkages stabilizing adjacent coatprotein dimers. The total energy applied to the system from thermal andshear stress sources was estimated and compared to the total energy ofdisulfide bonds present to validate this theory. The peak at 130° C.from the DSC thermogram was integrated to yield a total disulfide bondenergy of 43,860 kJ per mol of particle, assuming the peak centered at130° C. corresponded to disulfide bond breakage. Theoretical calculationof the total disulfide bond energy per particle using the bond enthalpyof a disulfide bond yielded a value of 45,180 kJ/mole, in good agreementwith the DSC result; further validating the peak assignment of 130° C.as disulfide bond breakage. Therefore, the amount of disulfide bondenergy in each sample was calculated using the value derived from theDSC peak integration and the amount of Qβ present in each sample. Theenergy derived from the shear and thermal effects during shearapplication was calculated and normalized by the disulfide bond energyper sample for comparison. The resulting plot clearly shows that theenergy contribution of the shear stress does not greatly affect thesystem until shear rates of 25 and 50 s⁻¹ (FIG. 6B). The thermal energypresent in the system is always 20% below the disulfide bond energy bythese calculations and remains constant for all samples. The shearenergy increase observed only with 25 and 50 s⁻¹ and subsequent increasein total applied energy relative to the total disulfide bond energy inthe system support the conclusion that the higher shear rates result indisulfide bond disassociation between dimers.

After the validation and analysis of the effect of processing conditionson VLP integrity, the effects of loading level and additives oncylindrical materials containing Qβ was studied to determine how Qβwould release from the implant in vitro. Understanding the releaseproperties in vitro was important in designing an optimal system for invivo implantation that would release appropriate amounts of VNP toelicit an immune response without excessive burst release phases orextremely slow release. All of the samples studied were manufactured viamelt-encapsulation with the syringe-die extrusion device and usedwithout any further post-processing. First, PEG additives were utilizedto determine the effect on release of Qβ from 1 wt % loaded samplesprepared via syringe-die melt-encapsulation. Samples loaded with 1 wt %Qβ did not demonstrate any burst release and had a significant lagperiod over the first 15 days (FIG. 7A). The first 15 days of releasefrom PLGA materials corresponds to the initial swelling and inductionphase, where the polymer matrix swells and minimal hydrolysis of thepolymer occurs. The lowest loading level of Qβ exhibited a significantdelay in release, likely due to the VLPs remaining within unswelledregions of PLGA until the matrix begin to degrade. Release begins after15 days as the polymer degrades and erodes, allowing for the Qβ todiffuse out of the matrix into the surrounding. This process continueduntil day 80 when the material had degraded into small pieces insolution. The total amount of protein released was -62% of the totalamount present. PEG additives were added during the melt-encapsulationprocess to accelerate the release, as PEG is a known porogen for PLGAmaterials. Upon hydration of the material, PEG will diffuse into theaqueous media rapidly leaving behind voids through which Qβ can diffuse.Two PEG molecular weights were used (8 and 20 kDa) to avoid negativeimmune responses in vivo and to keep the molecular weight of the porogenin the same range as PLGA. Both PEG molecular weights were manufacturedat 10 wt % loading levels and resulted in a burst release of Qβ duringthe initial swelling. The Qβ release was increased over the inductionphase from day 10 to 30 as the VLPs were able to diffuse more readilythrough the matrix as porosity was increased by PEG. Matrix erosionstarted after day 30 and the remaining Qβ was released rapidly as aresult of oligomeric PLGA species diffusing more rapidly from thematrix. No significant difference was observed between 8 and 20 kDa PEGadditive (FIG. 7A). PEG sizes from 10 to 20 kDa exhibit hydrodynamicradii of 3 to 3.5 nm, thus the small difference in hydrodynamic sizebetween 8 and 20 kDa PEG results in the minimal differences seen inrelease profiles. Nonetheless, either PEG additive greatly acceleratedthe release rate of Qβ and had no negative effect during processing.

Loading level is known to influence the release profile fromprotein-laden PLGA materials, thus the effect of loading for PLGAsamples containing 1, 5, and 10 wt % Qβ was studied. Increasing theloading level to 5 and 10 wt % Qβ increased the amount released over theswelling and induction phase by 10% compared to 1 wt % Qβ samples (FIG.7B). Furthermore, the release after the initial burst was relativelylinear for both loading levels. After matrix erosion started, therelease increased dramatically and all samples followed a similarrelease profile regardless of loading level. The increased loadinglevels of 5 and 10 wt % had little effect on the matrix erosion phase,which is hypothesized to be due to the small size of the VLPs notgreatly increasing the void size after diffusion out of the matrix. Thevoid size allowing for oligomeric PLGA diffusion would control the speedat which the matrix erodes, and the loading levels explored did notappear to affect this greatly enough to influence the overall releaseprofile. All samples broke down into small pieces in solution at 80 daysand had similar final cumulative release levels. FPLC analysis ofsamples collected at the 2 and 50-day time points released from implantsloaded with 10% Qβ indicated good stability throughout the releaseprocess with minimal increase in particle aggregation or break-up.

The in vitro release of 10 wt % Qβ loaded PLGA was studied in releasemedium with varying ionic strengths to determine how interparticle andparticle-polymer interactions effect release behavior. Increasing theionic strength by increasing the molarity of NaCl has previously beenshown to increase the release of lysozyme from PLGA microspheres throughdisruption of ionic interactions between carboxylic acid moieties inPLGA and the cationically charged lysozyme. The release of Qβ from thePLGA implants exhibited a clear dependence on ionic strength, withdecreasing amounts released in response to higher concentrations ofNaCl. Qβ exhibits a negative zeta potential at pH 7.4, indicating thatunder the release conditions the particles would exhibit an overallnegative charge. Therefore, increasing salt concentration would shieldthe negative charges on both Qβ and PLGA and decrease repulsion betweenboth adjacent Qβ particles and Qβ with PLGA. The decrease in ionicrepulsion due to charge shielding would result in closer association andaggregation between particles; a similar result has been previouslyobserved in surface adsorption studies of Qβ. The increase in ionicstrength has also been thought to slow release by decreasing PLGAswelling via charge shielding. We speculate this would result in a“jamming” effect of particles as they diffuse out of the polymer matrixthrough water filled pores and channels, slowing the release andresulting in the observed decrease in release rate with increasing ionicstrength.

The release samples at all ionic strengths were then incubatedsequentially with buffered solutions of 1 M NaCl, 5 M guanidinehydrochloride (GnHC1), and 5 mM sodium dodecyl sulfate (SDS) todetermine the factors resulting in the observed incomplete release ofQβ. The addition of 1 M NaCl did not result in any further significantrelease of Qβ, indicating that ionic interactions do not play a majorrole in unreleased Qβ, which is consistent with the decreasing releaseof Qβ in response to increasing ionic strength. Further incubation with5 M GnHCl, which would disrupt non-covalent aggregates of Qβ, resultedin an increase in released Qβ for all samples with higher ionic strengthsamples exhibiting higher amounts of released protein. This resultindicated that non-covalent aggregation of Qβ is a factor in particlesremaining entrapped within the polymer matrix and that high ionicstrength release medium results in more aggregation between particles.The final incubation with SDS would break-up any aggregates notdisrupted via GnHC1 incubation and Qβ adsorbed on PLGA. The results ofSDS incubation released an additional 42-53% of Qβ, indicating asignificant amount of Qβ remained within the polymer matrix due toadsorption onto the polymer. This amount was nearly the amount of Qβremaining from the previous release study and all samples reachedapproximately full cumulative release after incubation with SDS. Basedon this release study, the incomplete release of Qβ was predominatelydue to adsorption of Qβ onto the polymer. Non-covalent aggregates of Qβwere formed more readily with increasing ionic strength, based on therelease observed with GnHCl, owing to charge shielding. These factors inthe incomplete release of Qβ are common issues observed in the releaseof proteins from PLGA systems. While in vitro release studies areimportant to understand how the release of Qβ is influenced by differentfactors after melt-encapsulation, the behavior of PLGA materials in vivois much more complex due to multitude of enzymes, chemicals, and fluiddynamics present.

PLGA materials laden with Qβ were then utilized to assess theirperformance to stimulate a humoral immune response in a murine model; wetested whether a robust IgG response was generated against Qβ aftermelt-encapsulation and release. The subcutaneously implanted Qβ ladendevices were evaluated alongside a subcutaneous immunization schedule of50 μg Qβ injected 3 times biweekly (FIG. 8A). Control implantation ofneat PLGA cylinders indicated mild swelling over the first 2 weeks, withmice exhibiting no other adverse health or behavioral conditions. Theamounts injected and schedule were based on previous studies utilizingVLPs displaying antigen epitopes without adjuvants to successfullygenerate humoral immunity in mice. PLGA loaded with 10 wt % Qβ wasutilized for the immunization studies and mice were implanted with 0.5cm (˜8 mg) of 1 mm cylindrical material. The amount of implantedmaterial correlated to ˜150 μg of released Qβ over 30 days based on thein vitro release profile, delivering roughly the same amount of Qβ overthe first 28 days as the mice immunized via subcutaneous injection.Consistent levels of anti QP-IgG titers were observed over 65 days andthe booster administration lead to a successful increase in IgG levels,as expected. Overall, the Qβ vaccine implant matched the IgG titerprofile compared to a contemporary repeat-administration schedule usingsoluble Qβ. The resulting titers seen were also consistent with previousstudies using VLPs to immunize with the same vaccination schedule. Thisdemonstrates that the VLP delivery systems manufactured viamelt-encapsulation can potentially eliminate the need for multipleinjections for immunization and that the VLPs maintain the integrity ofthe surface epitopes after melt-encapsulation.

Different subtypes of IgG are indicative of different mechanisms ofimmune system activation, hence the subtypes of anti-Qβ IgG weredetermined at day 49, for both the implanted and injected animals (FIG.8B). In the future, the Q(3/PLGA platform is envisioned as a singleadministration cancer immunotherapy. Both immunization methods generatedpredominately IgG2a, which has a high binding ability of FCy receptorsand mediates the antibody dependent cell cytotoxicity (ADCC) of cancercells by neutrophils. This is important when exploring Qβ as a cancervaccine candidate, as this pathway is necessary to utilize the immuneresponse to prevent cancer. IgG1 was the second most predominate speciesfor both immunization methods and is involved in complement fixation andthe ADCC by natural killer cells. IgG2b was a minor fraction of both IgGpools and serves a similar function as IgG2a. The ability ofmelt-encapsulated Qβ to generate a statistically identical IgG subtypeprofile when compared to injected Qβ further validated the implantedmaterial as an alternative delivery vehicle for VLPs.

Melt-encapsulation was a viable method to create polymeric materialsladen with Qβ particles. Qβ maintained integrity with minimalaggregation after processing at 95° C. and withstood emulatedpost-processing conditions of compression molding andextrusion/injection molding. The relationship of Qβ aggregation to shearrate was non-dimensionalized to be applicable to other polymer systemsand processing conditions. The addition of PEG and increasing loadinglevel increased the amount of Qβ released over time and the materialsprepared were able to sustain Qβ delivery over an 80 day period invitro. PLGA materials loaded with 10 wt % Qβ were able to generate thesame levels of anti-Qβ IgG relative to a 3 injection immunizationschedule in vivo. Furthermore, the IgG subclass types generated werepresent in the same percentages between mice immunized via implantationor injection. The IgG subclasses generated are identical to injectionswith boosts, providing confidence that the Qβ delivery system can beexpanded to include a variety of vaccination targets. These resultsdemonstrate that VLPs can be successfully melt-encapsulated with PLGAand maintain structural integrity and biochemical signature to affectthe immune system in vivo.

EXAMPLE 2 Polymeric Microneedle Arrays

In Example 1, the viral nanoparticle Qβ was successfully incorporatedinto PLGA materials via melt processing and was effective as a singleadministration vaccine device. Anti-Qβ antibodies were generated afterimplantation of the Qβ/PLGA material and the subtypes of IgG indicatedthe immune response was the same as that of mice immunized with repeatadministrations of Qβ solutions. While Qβ/PLGA material was effectivefor vaccination, the implantation of solid polymeric materials can beinvasive and difficult in a clinical setting. One solution to thisadministration limitation with materials containing vaccines ismicroneedle arrays that administer vaccines through the skin. Theadministration of vaccines into the skin is highly effective due to thedermis containing dendritic cells, keratinocytes, T-lymphocytes,leukocytes, and a multitude of other cells necessary for mediation of anadaptive immune response. Transdermal administration of vaccines hasbeen attempted, however it is limited to hydrophobic low molecularweight antigens that can cross the epithelial layer.

To overcome this, micron-sized needles that penetrate through the outerlayer of skin into the dermis were developed. These microneedles rangedin the size of 50 to 1000 μm in length and can be conical or pyramidalin shape with diameters as small as 1 μm. We developed biodegradablePLGA based dissolving microneedle arrays due to PLGA being used in FDAdevices and serving as the basis for several developed microneedlesystems. Many vaccines are unable to withstand the temperature and timerequired for melt molding; however, we have previously shown that theviral nanoparticle Qβ can withstand temperatures necessary for meltmolding with PLGA and effectively serve as a vaccination agent uponrelease. Therefore, PLGA/Qβ composites that were prepared via meltprocessing were further melt molded using silicone microneedle moldsinto microneedle arrays. The aggregation state, biochemical signature,and ability of Qβ to be administered in a porcine skin puncture modelafter molding into a microneedle array were determined as well as themorphology and strength of the microneedle array.

Materials and Methods Materials

Poly(lactic-co-glycolic acid) (EXPANSORB® 10P019, 50:50 PLGA, inherentviscosity 0.15-0.25 dlg⁻¹, 5-20 kDa) was purchased from PCAS. Potassiumphosphate monobasic anhydrous, potassium phosphate dibasic anhydrous,sodium phosphate dibasic hetptahydrate, Gibco 1X PBS pH 7.4, butanol,Miller L B Broth, D-sucrose, sodium azide, sodium chloride, ethylacetate, PNPP tablets, Tween-20, albumin standard, chloroform, n-butanoland sodium hydroxide were purchased from Fisher Scientific.α-cyanohydroxycinnaminic acid was purchased from Sigma-Aldrich.Poly(ethylene glycol) (M_(n)=8000) was purchased from Amresco. Bradfordreagent was purchased from VWR. Dry milk was purchased fromLabScientific Inc. Uranyl acetate 2% solution was purchased fromElectron Microscopy Sciences. PLGA-FPI749 was purchased from Akina Inc.Goat anti-mouse IgG-alkaline phosphatase and were purchased from LifeTechnologies. Cy5-NHS dye was purchased from R&D systems. All reagentswere used directly, without further purification.

Instrumentation

Fast protein liquid chromatography (FPLC) was performed using a GEHealthcare AKTA-FPLC 900 chromatography system equipped with a Sephacryl1000 SF 10/300 size exclusion column. For all FPLC experiments, themobile phase was 50 mM phosphate buffer, with 150 mM NaCl (pH 7.4) at aflow rate of 0.4 mL/min. Samples were injected at a concentration of0.1-0.75 mg/mL and the resulting chromatograms were normalized by themaximum absorbance at 260 nm. Dynamic light scattering (DLS) experimentswere performed on a Wyatt DynaPro NanoStar DLS instrument. Samples wereanalyzed at 25° C. in plastic disposable cuvettes with a path length of10 mm. Transmission electron microscopy (TEM) was performed on a FEITechnai TF30 ST microscope. Negative stained TEM samples were mounted on400 mesh hexagonal copper grids bearing Formvar support film, stainedwith 2% uranyl acetate solution, and allowed to dry for 12 h. Microplatemeasurements were taken with a Biotek Synergy HT microplate reader.Centrifugation was performed with an Eppendorf 5424 centrifuge.Ultracentrifugation was performed with a Beckman Coulter Optima L-100 XPultracentrifuge. Scanning electron microscopy was performed using aJEOL-6510LV scanning electron microscope at 1 kV. Compression testingwas performed using an MTS Insight Electromechanical Testing system withcompression attachments and a 5 kN load cell. Mass spectra werecollected using a Bruker Autoflex III MALDI-TOF-TOF mass spectrometerwith a 200 Hz Smartbeam II laser system and an a-cyanohydroxycinnaminicacid matrix. Fluorescent images of the porcine skin puncture site werecollected with a CRi Maestro fluorescent imaging system with a yellowfilter set (576-621 nm excitation, 635 low pass emission filter).Confocal images were collected using a Leica TCS SPE microscope with a635 nm solid state laser.

Qβ Expression and Purification

Qβ was prepared based on the protocol described in the previous Example.A frozen glycerol stock of chemically competent BL21(DE3) E. coli cellstransformed with pET28CP (containing the Qβ coat protein sequence) inlysogeny broth (LB) media containing kanamycin (50m/mL) was thawed and 1μL was added to 100 mL of autoclaved selective LB media and grown tosaturation for 12 h at 37° C. A total of 10 mL of culture was thendiluted into 1000 mL of freshly prepared selective LB media. Culturegrowth was monitored by optical density at 600 nm (OD600). When theOD600 of the cultures reached approximately 0.8 (mid log phase), proteinexpression was induced with the addition of 10 mL of 100 mM IPTG, givinga final IPTG concentration of 1 mM. Shaking was continued at 37° C. foran additional 6 h, at which point cells were collected by centrifugationin an Eppendorf A-4-81 rotor at 4000 rpm (4° C.) for 30 min. Thesupernatant was decanted, and the cell pellet was frozen at −80° C.until purification. Cells were then resuspended in ˜100 mL of PBS, pH7.4. The buffer used for the original resuspension continued to be usedfor subsequent steps of particle preparation. Samples were chilled onice and then sonicated with a probe sonicator (10 min total sonicationtime, 5 s on and 5 s off, 60-70 W power output) in an ice bath to lysecells. The cell debris was pelleted in an Eppendorf FA-45-6-30 rotor at10000 rpm for 10 min, and the supernatant was decanted and collected.The Qβ particles were precipitated from the resulting supernatant by theaddition of 10% w/v PEG8000 at 4° C. for 12 h on a rotisserie. Theprecipitated fraction was isolated from the supernatant bycentrifugation in an Eppendorf FA-45-6-30 rotor for 10 min (4° C.) at10,000 rpm. The pellet was redissolved in ˜20 mL of PBS and extractedwith a 1:1 v/v solution of n-BuOH/CHCl₃ to remove excess lipid. Theaqueous fraction was collected following centrifugation using aFA-45-6-30 rotor for 10 min, 4° C. at 10000 rpm. Qβ particles werepurified on 10-40% sucrose gradients in an SW28 rotor at 28000 rpm for 4hours. Approximately 4 mL of light scattering Qβ solution was pulledfrom each gradient tube and subsequently pelleted in an ultracentrifuge(50.2Ti rotor, 42K, 3 h). The purified Qβ particles were dissolved inPBS (pH 7.4) and purity was verified via FPLC and DLS. A liter culturetypically yielded ˜100 mg of pure Qβ. Qβ particles were spin-filteredinto deionized water using 100 kDa MWCO spin filters and frozen. Thesamples were then lyophilized for 3 days to yields a solid white powder.

Melt Processing of Qβ

Poly(lactic-co-glycolic acid) (PLGA) was individually ground manuallywith a mortar and pestle twice, 10 minutes each time, into a finepowder. PLGA was mixed with the 10 weight percent of lyophilized Qβ viarepeated vortexing in a 2 mL Eppendorf tube. A custom built aluminumsyringe-die were used for melt processing of the blends to minimizematerial input. The syringe-die systems consisted of a cylinder with acircular 1 mm exit diameter that was wrapped with heating tape, combinedwith a digital control element to provide constant heating. The cylinderwas designed to fit a polypropylene 1 mL volume Norm-Ject syringe whichwere filled with 300-350 mg of the PLGA/Qβ blend. The blend was heatedat 95° C. as determined by a glass thermometer for 10 minutes. Themelted PLGA/Qβ blend was flowed through the die using a syringe pumpwith a velocity of 3 mm s⁻¹ (˜2.35 mm³ s⁻¹ volumetric flow rate). Theresulting cylindrical implants had diameters ranging from 1.0-1.3 mm.

Qβ Dye Conjugation

Qβ was conjugated with fluorescent N-hydroxysuccinimide functionalizedCy5 dye through reaction with amines from lysine residues on the surfaceof Qβ, 15 mg of Qβ in 2.5 mL of phosphate buffer (100 mM, pH 8) wasadded to two amber 1.5 mL Eppendorf tubes, with 1.25 mL of Qβ solutionin each tube. 400 molar equivalents of NHS-Cy5 in DMSO (1.46 mg in 324μL total volume, 162 μL per tube) were added to the Qβ solutions,vortexted, and incubated at room temperature for 4 hours with rotaryagitation at 100 rpm. The excess dye was removed via repeatedcentrifugal filtration using 100K MWCO spin filters until no absorbanceat 650 nm, indicative of Cy5 dye, was detected via UV-vis spectroscopy.The Qβ-Cy5 particles were analyzed via DLS and FPLC with wavelengthsmonitored at 280, 260, and 650 nm. The particles were then spin filteredinto deionized water and lyophilized. PLGA material laden with 10 wt %Qβ-Cy5 was prepared via the previously described method.

Microneedle Fabrication

Microneedle arrays were fabricated via melt molding utilizing siliconemolds (Micropoint Technologies) that were designed to yield an array of100 pyramidal needles with a base size of 100×100 μm and a length of 250μm. The microneedle molds were filled with ˜2 cm lengths of neat PLGA,10%Qβ/PLGA, or 10%Qβ-Cy5/PLGA material (˜120 mg) and incubated in avacuum oven at 95° C. The material was incubated in the oven undervacuum for 10 minutes, then vented to atmospheric pressure for 10minutes. This cycle was repeated a total of 3 times and the samples werethen removed and kept at ˜20° C. for 30 minutes.

The resulting microneedle arrays of were sputter coated with a 10 nmlayer of gold and imaged via SEM. The mechanical properties of theneedles were measured via compression testing with a rate of 10 μm/s andthe maximum strength of the needle was determined from the force valueat saturation. The values were reported as the average and standarddeviation of 3 samples. Particles were recovered via ethyl acetateextraction performed by dissolving -100 mg of material in 1 mL of ethylacetate for 15 minutes. This was followed by centrifugation for 5minutes at 5,000 rpm using an Eppendorf 5810 R centrifuge with a fixedangle rotor, based on a previously established protocol for organicextraction of active lysozyme. The supernatant was decanted and theprocess was repeated two more times. The remaining solids were driedunder vacuum at room temperature for 24 hours. The solid proteinrecovered was resuspended in PBS for 24 hours at 4° C. and analyzed viaFPLC, DLS, and TEM.

Porcine Skin Puncture and Imaging

Porcine skin was a generous gift from the Dr. Minh Lam and theDepartment of Dermatology. Skin samples were collected from freshlysacrificed pigs and immediately stored at ˜80° C. The porcine skin wasremoved from ˜80° C. and allowed to thaw at room temperature. The hairwas shaved from the skin and a 10 wt % QP-Cy5 loaded PLGA microneedlearray was applied to the skin and affixed with a layer of parafilm incontact with the skin and tape to ensure the array stayed in place. Theskin was placed in an incubator at 37° C. with 95% relative humidity anda solid plastic block weighing 0.713 g was used to apply 7 N ofapplication force to the array for 1 hour. The weight was then removedand the skin with the affixed microneedle array was incubated for 48more hours. After incubation, the microneedle array was removed and boththe array and the application site on the skin were imaging via Maestrofluorescence imaging.

ELISA Analysis of Processed Qβ

ELISA was utilized in order to determine the retention of thebiochemical surface characteristic of Qβ after melt processing andmicroneedle molding. The melt processed and microneedle molded sampleswere recovered via ethyl acetate extraction and the concentration wasdetermined via Bradford assay. Nunc Maxisorp 96-well plates with 1 μg ofQβ sample in 200 μL of PBS, pH 7.4 at 4° C. overnight. The wells werethen blocked with 200 μL of blocking buffer (2.5% w/v dry milk in PBS,pH 7.4) at 37° C. for 1 hour. The wells were then incubated with a1:2500 dilution of mouse sera collected on day 28 from mice that hadbeen immunized with 3 injections of 50 μg of Qβ on a biweekly basis. Thewells were then incubated with 100 i.iL of a 1:1000 dilution in blockingbuffer of alkaline-phosphatase labeled goat anti-mouse IgG for 1 hour at37° C. The wells were washed between each incubation step using 3×200 μLof 0.1% w/v Tween-20 in PBS, pH 7.4. The wells were developed using 100μL of PNPP substrate tablets (1 mg/mL), dissolved in 0.1 M glycinebuffer at pH 10.4, at 4° C. for 30 minutes. The reaction was stoppedwith 100 μL of 2 M NaOH and the absorbance was read at 405 nm intriplicate for each sample. Values are expressed as the average andstandard deviation of measurements using sera from 2 mice.

Qβ Chimeric Particle Design and Production

The P4 and CH401(Rat) amino acid sequences were inserted at theC-terminal of the Qβ coat protein. A flexible serine and glycine linkerwas added between the coat protein and peptide to allow the peptide tobe displayed effectively. The amino acid sequences are shown below withthe linker highlighted in blue.

P4: (SEQ ID NO: 1) GGSGSGGPESFDGDPASNTAPLQPEQLQ CH401(Rat):(SEQ ID NO: 2) GGSGSGGYQDMVLWKDVFRKNNQLAP

The DNA coding for the amino acid sequences was optimized for E. colicodon usage using JCat software. The DNA sequence for Qβ coat protein-P4was synthesized using primer overlap PCR with Ncol and Xhol cutsites atthe 5′ and 3′ ends respectively. The DNA sequence for Qβ coatprotein-CH401 was synthesized by GenScript with Ncol and Xhol cutsitesat the 5′ and 3′ ends respectively. The DNA sequences are shown belowwith the Ncol/Xhol cutsites highlighted in red and the peptide sequenceshighlighted in green.

P4: (SEQ ID NO: 3) GATATACCATGGCAAAATTAGAGACTGTTACTTTAGGTAACATCGGGAAAGATGGAAAACAAACTCTGGTCCTCAATCCGCGTGGGGTAAATCCCACTAACGGCGTTGCCTCGCTTTCACAAGCGGGTGCAGTTCCTGCGCTGGAGAAGCGTGTTACCGTTTCGGTATCTCAGCCTTCTCGCAATCGTAAGAACTACAAGGTCCAGGTTAAGATCCAGAACCCGACCGCTTGCACTGCAAACGGTTCTTGTGACCCATCCGTTACTCGCCAGGCATATGCTGACGTGACCTTTTCGTTCACGCAGTATAGTACCGATGAGGAACGAGCTTTTGTTCGTACAGAGCTTGCTGCTCTGCTCGCTAGTCCTCTGCTGATCGATGCTATTGATCAGCTGAACCCAGCGTATCTGGTGGTCCGGAATCTTTCGACGGTGACCCGGCTTCTAACACCGCTCCGCTGCAGCCGGAACAGCT (SEQ ID NO: 4GCAGTAATAAGGATGACTCGAGTCTGGCTGCA CH401 (Rat): (SEQ ID NO: 5)GATATACCATGGCAAAATTAGAGACTGTTACTTTAGGTAACATCGGGAAAGATGGAAAACAAACTCTGGTCCTCAATCCGCGTGGGGTAAATCCCACTAACGGCGTTGCCTCGCTTTCACAAGCGGGTGCAGTTCCTGCGCTGGAGAAGCGTGTTACCGTTTCGGTATCTCAGCCTTCTCGCAATCGTAAGAACTACAAGGTCCAGGTTAAGATCCAGAACCCGACCGCTTGCACTGCAAACGGTTCTTGTGACCCATCCGTTACTCGCCAGGCATATGCTGACGTGACCTTTTCGTTCACGCAGTATAGTACCGATGAGGAACGAGCTTTTGTTCGTACAGAGCTTGCTGCTCTGCTCGCTAGTCCTCTGCTGATCGATGCTATTGATCAGCTGAACCCAGCGTATGGTGGTTCTGGTTCTGGTGGTTACCAGGACATGGTTCTGTGGAAAGACGTTTTCCGTAAAAACAACCAGCTGGCTCCGTAATAAGGATGACTCGAGTCTGGCTGCA

Both DNA sequences and pCDF expression vector were double digested withNcol and Xhol and agarose gel band purified. The digested DNA sequenceswere then individually ligated with the digested pCDF vector, ligated,transformed into NEB5a chemically competent cells, and plated ontospectinomycin containing selective LB medium agar plates. The plasmidwas purified from an individual colony and successful ligation wasverified via sequencing. The pCDF-QβP4 or pCDF-QβCH401(Rat) plasmid wasco-transformed with pET28 expression vector containing the wild type Qβ(pET28-Qβ coat protein into ClearColi® BL21(DE3) E. coli (Lucigen) viaelectroporation. The transformed E. coli were plated onto spectinomycinand kanamycin containing selective LB medium agar plates.

The chimeric particles were prepared based on a modified protocoldescribed previously. A single colony from plated ClearColi® BL21 E.coli containing either pCDF-QβP4/pET28-Qβ or pCDF-QβCH401(Rat)/pET28-Qβwas added to 100 mL of autoclaved selective containing spectinomycin andkanamycin (50 μg/mL for both antibiotics) LB media and grown tosaturation for 12 h at 37° C. A total of 10 mL of culture was thendiluted into 1000 mL of freshly prepared selective LB media. Culturegrowth was monitored by optical density at 600 nm (OD600). When theOD600 of the cultures reached approximately 0.8 (mid log phase), proteinexpression was induced with the addition of 10 mL of 100 mM IPTG, givinga final IPTG concentration of 1 mM. The temperature was then lowered to30° C. and incubated at 37° C. for an additional 15 h, at which pointcells were collected by centrifugation in an Eppendorf A-4-81 rotor at4000 rpm (4° C.) for 30 min. The supernatant was decanted, and the cellpellet was frozen at ˜80° C. until purification. Cells were thenresuspended in ˜100 mL of PBS, pH 7.4. The buffer used for the originalresuspension continued to be used for subsequent steps of particlepreparation. Samples were chilled on ice and then sonicated with a probesonicator (10 min total sonication time, 5 s on and 5 s off, 60-70 Wpower output) in an ice bath to lyse cells. The cell debris was pelletedin an Eppendorf FA-45-6-30 rotor at 10000 rpm for 10 min, and thesupernatant was decanted and collected. The Qβ particles wereprecipitated from the resulting supernatant by the addition of 10% w/vPEG8000 at 4° C. for 12 h on a rotisserie. The precipitated fraction wasisolated from the supernatant by centrifugation in an EppendorfFA-45-6-30 rotor for 10 min (4° C.) at 10,000 rpm. The pellet wasredissolved in ˜20 mL of PBS and extracted with a 1:1 v/v solution ofn-BuOH/CHC13 to remove excess lipid. The aqueous fraction was collectedfollowing centrifugation using a FA-45-6-30 rotor for 10 min, 4° C. at10000 rpm. The particles were purified on 10-40% sucrose gradients in anSW28 rotor at 28000 rpm for 4 hours. Approximately 4 mL of lightscattering particle solution was pulled from each gradient tube andsubsequently pelleted in an ultracentrifuge (50.2Ti rotor, 42K, 3 h).The purified particles were dissolved in PBS (pH 7.4) and purity wasverified via FPLC, DLS, and TEM. The amount of peptide bearing coatprotein was determined via SDS-PAGE and MALDI-TOF spectroscopy. TheSDS-PAGE result was analyzed via pixel density analysis using ImageJsoftware and the MALDI-TOF result was analyzed via peak integration. Aliter culture typically yielded ˜50 mg of pure particle. For meltprocessing, the particles were spin-filtered into deionized water using100 kDa MWCO spin filters and frozen. The samples were then lyophilizedfor 3 days to yields a solid white powder.

Qβ Chimeric Particle In Vivo Studies

All experiments were carried out in accordance with Case Western ReserveUniversity's Institutional Animal Care and Use Committee. Male Balb/cmice (Charles River) aged 7 weeks (n=5 for each treatment group) wereinjected 3 times on days 0, 14, and 28. The mice were injected witheither 50 μg of Qβ, Qβ-P4, or Qβ-CH401 or 2 μg of P4 or CH401 freepeptide in 100 μL sterile PBS through subcutaneous injections behind theneck using a 29G insulin syringe. The amount of free peptide injectedwas the amount of peptide displayed on the chimeric particle calculatedusing the SDS-PAGE and MALDI result. All Qβ samples were produced inClearColi E. coli cells that contain a modified lipopolysaccharide (LPS)outer membrane. Blood (-100 (IL) was drawn prior to the firstimmunization and on a biweekly basis via the retro-orbital plexus usingheparinized capillary tubes and collected in Greiner Bio-One VACUETTE™MiniCollect™ tubes. Serum was separated by centrifuging blood samples at14,800 rpm, 4° C., for 10 min and stored at 4° C. until analyzed viaenzyme-linked immunosorbent-assay (ELISA).

The anti-Qβ IgG response was measured by first coating Nunc Maxisorp96-well plates with 1 μg of Qβ in 200 (IL of sterile PBS, pH 7.4 at 4°C. overnight. The wells were then blocked with 200 (IL of blockingbuffer (2.5% w/v dry milk in PBS, pH 7.4) at 37° C. for 1 hour. Thewells were then incubated with mouse sera at 1:500, 1:2500, and 1:12500dilutions in 100 (IL blocking buffer for 2 hours at 37° C. The wellswere then incubated with 100 μL of a 1:1000 dilution in blocking bufferof alkaline-phosphatase labeled goat anti-mouse IgG for 1 hour at 37° C.The wells were washed three times between each incubation step using 200(IL of 0.1% w/v Tween-20 in PBS, pH 7.4. The wells were developed using100 (IL of PNPP substrate tablets (1 mg/mL), dissolved in 0.1 M glycinebuffer at pH 10.4, at 4° C. for 30 minutes. The reaction was stoppedwith 100 (IL of 2 M NaOH and the absorbance was read at 405 nm intriplicate for each sample. Values are expressed as the average andstandard deviation of 5 mice.

The anti-P4 or anti-CH401 IgG response was measured by coating PierceMaleimide Activated 96-well plates with 0.2 (ig of peptide in 200 μL ofsterile PBS, pH 7.4 with 10 mM EDTA overnight at 4° C. The wells werethen blocked with 100 μL of 10 (Ig/mL L-cysteine solution in PBS with 10mM EDTA for 1 hour at 37° C. The wells were then incubated with mousesera at 1:500, 1:2500, and 1:12500 dilutions in 100 μL of PBS with 10 mMEDTA for 2 hours at 37° C. The wells were then incubated with 100 (IL ofa 1:1000 dilution in PBS with 10 mM EDTA of alkaline-phosphatase labeledgoat anti-mouse IgG for 1 hour at 37° C. The wells were washed threetimes between each incubation step using 200 (IL of 0.1% w/v Tween-20 inPBS, pH 7.4. The wells were developed using 100 (IL of PNPP substratetablets (1 mg/mL), dissolved in 0.1 M glycine buffer at pH 10.4, at 4°C. for 30 minutes. The reaction was stopped with 100 μL of 2 M NaOH andthe absorbance was read at 405 nm in triplicate for each sample. Valuesare expressed as the average and standard deviation of 5 mice.

Qβ Chimeric Particle Melt Processing

Poly(lactic-co-glycolic acid) (PLGA) was individually ground manuallywith a mortar and pestle twice, 10 minutes each time, into a finepowder. PLGA was mixed with the 10 weight percent of lyophilized Qβ,Qβ-P4, or Qβ-CH401(Rat) via repeated vortexing in a 2 mL Eppendorf tube.A custom built aluminum syringe-die were used for melt processing of theblends to minimize material input. The syringe-die systems consisted ofa cylinder with a circular 1 mm exit diameter that was wrapped withheating tape, combined with a digital control element to provideconstant heating. The cylinder was designed to fit a polypropylene 1 mLvolume Norm-Ject syringe which were filled with 150-200 mg of thePLGA/Qβ blend. The blend was heated at 95° C., as determined by a glassthermometer, for 10 minutes. The melted PLGA/Qβ blend was flowed throughthe die using a syringe pump with a velocity of 3 mm s⁻¹ (˜2.35 mm³ s⁻¹volumetric flow rate). The resulting cylindrical implants had diametersranging from 1.0-1.3 mm. Particles were recovered via ethyl acetateextraction performed by dissolving ˜100 mg of material in 1 mL of ethylacetate for 15 minutes. This was followed by centrifugation for 5minutes at 5,000 rpm using an Eppendorf 5810 R centrifuge with a fixedangle rotor, based on a previously established protocol for organicextraction of active lysozyme. The supernatant was decanted and theprocess was repeated two more times. The remaining solids were driedunder vacuum at room temperature for 24 hours. The solid proteinrecovered was resuspended in PBS for 24 hours at 4° C. and analyzed viaFPLC, DLS, and TEM.

Qβ Chimera Release Study

Release studies were conducted on samples of the melt processed implants(˜1 cm long, 10-15 mg, n=3). Samples were placed in 2 mL Eppendorf tubeswith 250 μL of Gibco 1×PBS with 0.01 wt % sodium azide and incubated at37° C. with 90% relative humidity. Aliquots of 225 μL were removed ateach time point and replaced with fresh buffer. The proteinconcentration at each time point was determined via Bradford assay withcomparison to a freshly prepared bovine serum albumin standard curve.

Results PLGA Microneedle Production and Characterization

Microneedles were produced using PLGA via a melt molding process wherethe polymer was melted into a silicone mold designed to yield a 10×10assembly of 250 μm long pyramidal needles. Melt molding was utilized asit does not require the long drying times or repeated application ofsolutions associated with coated and layer-by-layer assembly microneedlearrays. The PLGA was melted at 95° C. in a vacuum oven, with thetemperature chosen to represent the processing temperature previouslyused to melt process Qβ with PLGA, and subjected to 3 cycles ofdegassing to remove air bubbles in the polymer melt. The resultingmicroneedle array was imaged using SEM and exhibited needles of thecorrect size and shape based on the silicone mold design (FIG. 9A).Mechanical analysis of the needle strength via compression testingindicated the needles had a maximum strength of 0.349 ±0.0572 N perneedle and an overall maximum strength of 34.9 ±5.72 N for the totalarray (FIG. 9B). These ultimate strength values are in the range ofpuncture strength values for previous microneedle skin application,indicating the molded PLGA microneedle arrays were suitable for dermaladministration.

Qβ/PLGA Microneedle Production

After verification that PLGA microneedle arrays could successfully befabricated at the temperature used for Qβ melt processing, microneedlearrays were prepared with PLGA containing 10 wt % Qβ. The PLGA materialcontaining 10 wt % Qβ was prepared using a syringe extrusion device at95° C. with a 10 minute incubation time. After melt processing withPLGA, the Qβ/PLGA material was melt molded into microneedle arraysfollowing the same procedure used to make PLGA arrays. The resultingarray exhibited similar needle morphology and had a maximum strength of0.333 ±0.0388 N per needle, indicating the incorportation of 10 wt % Qβhad negligible effect on the formation and strength of the microneedlesformed during melt molding.

The melt molding process subjected the Qβ to further heat and mechanicalstresses during the melting and degassing process. Qβ was recovered fromthe microneedle array via ethyl acetate extraction using the methoddescribed in the previous Example. Analysis of the recovered Qβ via FPLCindicated an increase in the aggregated species relative to initial meltprocessing studied previously (FIG. 10A). Curve fitting and integrationof the two major peaks in the FPLC chromatogram yielded relativepercentage of 14.1% for the aggregate peak at 10 mL and 85.9% for themajor peak centered at 18.5 mL corresponding to intact particles. DLSanalysis of the microneedle processed Qβ also indicated the presence ofaggregated species, with two major peaks at 24.6 and 118.7 nm (FIG.10B). These peaks had relative percentages of 75.4% and 24.6%respectively, with the aggregated species having a higher relativepercentage than that calculated from the FPLC result. This discrepancywas due to the limitations of the curve fitting process not fully takinginto account the peak broadening towards higher elution volumes of thepeak centered at 18.5 mL. The broadening was due to smaller aggregatedspecies in the sample that eluted between the main Qβ peak and the voidvolume. These aggregated species were included in both peaks of the DLSresult, resulting in the increase in value of the average radius for thelower peak from 15.1 nm to 24.6 nm and the higher percentage of thelarger peak relative to the FPLC result. TEM analysis of the recoveredQβ also verified the presence of intact viral nanoparticles, inagreement with the FPLC and DLS result (FIG. 10C). Overall, further meltprocessing with PLGA into microneedle arrays resulted in Qβ that had amajority of the population as single nanoparticles. The results seenwere similar to Q(3/PLGA material that was melt pressed, where Qβrecovered from the melt pressed samples exhibited an increase in theaggregated population in response to post-processing with further heatand pressure. The melt molding process subjected Qβ to the same stresseswith, further applied heat and mechanical stresses generated during thedegassing process where bubbles were forced out of the polymer melt.

While Qβ was able to be successfully recovered from the microneedlearray and maintain particle integrity, we sought to further explore theintegrity of the surface epitopes of Qβ after initial melt processingand melt molding into microneedle arrays. We have shown that miceimplanted with Q(3/PLGA devices were able to be immunized, therefore weexpected that the surface epitopes would be maintained after meltprocessing and microneedle production. ELISA was utilized with anti-QβIgG from sera that was generated in mice immunized with 50 μg doses ofQβ in solution following a standard 3, biweekly injection schedule. Qβ,Qβ that was processed at 10 wt % with PLGA and recovered, and 10 wt %Qβ/PLGA that was further melt molded into microneedles and recoveredwere all coated onto adsorbing ELISA plates and analyzed using immunizedsera from two mice. The results were normalized via the unprocessed QβELISA response to yield percent antibody recognition values (FIG. 11).The results indicated a small loss in antibody recognition for meltprocessed and microneedle molded Qβ, with percent recognition values of92.1 and 90.2% respectively. This loss in antibody recognition may bedue to particle aggregates blocking antibody binding sites ordegradation of surface residues through oxidation or chemical reactionwith other residues or PLGA. Both processed Qβ samples exhibited highererror in antibody recognition relative to native Qβ. This waspotentially due to inconsistent coating of the Qβ on the ELISA wellsurface by aggregated species between wells resulting in the obscuringof adjacent particles depending on the orientation of aggregates whenthey adsorb onto the surface. Overall, the ELISA result indicated thatmelt processing and microneedle processing did not result in a largeloss of the surface biochemical character of Qβ and that the thermal andmechanical stresses applied during microneedle molding does not havefurther impact after initial melt processing.

Porcine Skin Puncture Model

A porcine skin puncture model was used to determine the in vitro abilityof the microneedle arrays to effectively penetrate the skin and releaseQβ. The porcine skin puncture model is a commonly used in vitro modelfor skin puncture, as it is representative of the inhomogeneity of skinand hair follicle spacing. Qβ was conjugated with NHS functionalized Cy5fluorescent dye in order to visualize the location of Qβ after releaseinto the skin. FPLC characterization of Qβ-Cy5 indicated successfulattachment of Cy5 through co-localization of the Qβ peak with a peak at650 nm, the maximum absorbance of Cy5 (FIG. 12A). There was also no 650nm peak in the 30 to 35 mL elution volume range that would correspond tofree Cy5 dye, indicating that all excess dye had been removed. The DLShistogram of Qβ-Cy5 indicated the conjugation did not result in anyparticle aggregates (FIG. 12B).

The Qβ-Cy5 particles were then lyophilized, melt processed at 10 wt %with PLGA at 95° C., and melt molded into microneedle arrays via themethod previously described. The microneedle arrays containing Qβ-Cy5were fluorescent after melt processing and molding, indicating exposureto high temperatures during the processing did not degrade the Cy5 dye.The microneedle array was applied to shaved porcine skin using 7 N ofapplication force for 1 hour at 37° C., then the insertion force wasremoved and the microneedle patch was affixed with tape where it wasinserted for 48 hours. The insertion force of 7 N was chosen to be onthe lower end of insertion forces typically used in in vitro studies(10-20 N) to ensure the array did not fracture during administration.Clinical self-administration of microneedle patches usually involved themicroneedle in a cloth or plastic enclosure inserted via force from thefingers, which was typically 9-11 N for the entire array.

The patch was removed from the skin and puncture marks were clearlyvisible in the administration site. The microneedle array had no needlespresent on the surface, indicating they dissolved during theadministration process. The administration site was imaged optically andfluorescently (FIG. 13) and clearly indicated that the fluorescentlylabeled viral nanoparticles were released into the skin and diffused outthrough the puncture site. The area of diffusion was limited due to lackof fluid flow through the skin. This limitation can be overcome throughthe use of a Franz cell to mimic physiological fluid flow and enhancediffusion through the skin. The fluorescent image indicated areas ofhigh signal intensity around the left hand side of the administrationsite, corresponding to a higher density of hair follicles. Previousresults administering nanoparticles using microneedle arrays haveindicated they migrate to and accumulate in hair follicles, thus theQβ-Cy5 nanoparticles had a tendency to accumulate in hair follicles uponmicroneedle administration.

Chimeric Qβ-Her2 Epitope Chimeric Particle Design and Production

Qβ was further expanded as a vaccine platform through the display ofHER2+breast cancer epitopes on the nanoparticle surface. Passiveimmunotherapy utilizing a monoclonal antibody against HER2, herceptin,has seen great success in clinical application. HER2 epitope vaccinesare a promising platform for cancer immunotherapy to generate antibodiesagainst HER2 presenting cancer cells using a patient's own immune systemand eliminating the need for repeated injections of herceptin. There hasbeen extensive research into peptide based HER2 vaccines, however as aself-antigen present in the body that is normally tolerated there is aninherent hurdle into inducing a memory immune response against theepitope. This limitation can be overcome by presentation on viralnanoparticle systems to induce a strong immune response against thepresented antigen. Indeed, presentation of the peptide epitopes P4 andCH401 have been shown to induce a strong immune response against thepeptides in murine models. We sought to incorporate the P4 andCH401(Rat), a derivative of the human CH401 peptide sequence using therat amino sequence, onto the surface of Qβ through a genetic fusionstrategy. Genetic fusions of Qβ with peptides and proteins have beensuccessfully produced and utilized for vaccination and cell targeting.These have been accomplished through the addition of the peptidesequence to the C-terminal of the Qβ coat protein, which is exposed onthe surface of the assembled Qβ particle.

The DNA sequence of the Qβ coat protein was altered to include DNAcoding for a short, flexible linker consisting of glycine and serinefollowed by the P4 or CH401 sequence at the C-terminal of the Qβ coatprotein. The altered Qβ coat protein DNA sequence was then cloned intothe pCDF expression vector and transformed into E. coli simultaneouslywith a pET28 expression vector coding for unaltered Qβ coat protein.Expression of both the coat protein with the peptide extension and theunaltered coat protein allowed for the self-assembly of Qβ particlesconsistent of native coat protein and coat protein displaying thepeptide on the surface (FIG. 14).

Particles bearing either P4 or CH401(Rat) were successfully createdthrough the co-expression of both expression vectors and purified fromE. coli. The amount of peptide bearing coat proteins incorporated intoparticles was determined from both SDS-PAGE and MALDI-TOF analysis (FIG.15). The MALDI analysis had the appearance of peaks corresponding to theexpected molecular weights of coat protein fused to either peptide thatwere not present in wild type Qβ, further indicating the successfulincorporation of the peptide sequences into the chimeric particles. Theresults indicated that ˜32 P4 and ˜32 CH401(Rat) peptides wereincorporated into the chimeric Qβ particles respectively.

This amount of peptide incorporation was similar to the results observedwith chemical conjugation of the peptides to a viral nanoparticle ofsimilar size to Qβ. FPLC, DLS, and TEM analysis of the chimericparticles indicated that neither peptide induced particle aggregationand that the assembled particles exhibited the same size and morphologyas native Qβ particles (FIG. 16).

Chimeric Particle In vivo Vaccination

Following the successful expression and purification of P4 or CH401(Rat)peptide bearing chimeric particles, the ability of the particles toelicit a P4 or CH401(Rat) specific immune response was assessed. Micewere immunized via 3 repeated injections on a biweekly of either 50 μgof wild type Qβ, Qβ-P4, or Qβ-CH401(Rat), based on a previous viralnanoparticle conjugate vaccination study (FIG. 17). Two control groupswere injected with 2 μg of free P4 or CH401(Rat) peptide, correspondingto the amount of peptide displayed on 50 μg of Qβ-P4 or Qβ-CH401(Rat).Sera was collected from the mice to assess the immune response of themice in response to the Qβ carrier and the peptides.

The immune response of the treatment groups to the HER2 peptide and theQβ carrier was assessed via ELISA analysis of the collected sera fromday 0, 14, 21, and 28. The sera was tested against plates coated withthe P4 or CH401(Rat) peptide or plates coated with wild type Qβ,allowing for the separation of the immune response to both components ofthe chimeric particles. Analysis of the immune response to seracollected on day 0, prior to immunization, had no apparent response tothe peptides or Qβ as expected. Further ELISA analysis of the responseon days 14, 28 and 42 had exhibited no strong response to either the P4or CH401(Rat) peptide displayed on the surface or the chimeric particles(FIG. 18).

Mice treated with the free peptide also did not display an immuneresponse, likely due to the rapid clearance of the small peptide onceinjected, consistent with previous results. Treatment groups injectedwith Qβ or the chimeric Qβ particles did exhibit a strong immuneresponse to the Qβ carrier, demonstrating that the particles wereprocessed and presented by APCs to generate anti-Qβ antibodies (FIG.19).

The Qβ-P4 treatment group did exhibit some P4 peptide specific antibodyresponse at day 28 and 42, however the response was an order ofmagnitude lower than mice immunized with viral nanoparticles bearing P4via chemical conjugation. The lower response observed with the geneticfusion of the peptide to the coat protein versus chemical conjugationmay be due to differences in how the coat protein is processed once itwas taken up by antigen presenting cells (APCs). A short, flexible PEGlinker was utilized to couple the peptide to the virus for the chemicalconjugation prepared conjugates using a maleimide linkage between thePEG and the peptide. This maleimide linker is generally stable underphysiological conditions, however during the endosomal trafficking andprocessing there are typically high concentrations of glutathione andother reducing agents than can reduce the thio-ether linkage between PEGand the peptide. Processing of antigens by antigen presenting cellsinvolves extensive protease activity, thus the absence of the chemicallinker between the peptide and coat protein in the chimeric particle haymay have led to proteolytic cleavage of the presented peptide sequence,resulting in the low peptide specific antibody generation observed. Forfuture studies, altering the peptide linker sequence between the coatprotein and the peptide on the chimeric particles to one that is morereadily and selectively cleaved during endosomal processing may helpenhance the immune response by diminishing non-specific proteolyticcleavage of the presented peptide.

Melt Processing of Chimeric Qβ Particles

Despite the low peptide specific immunogenicity of the chimericparticles, we sought to determine whether incorporation of the peptidegenetic fusions into the particles had a negative impact on thestability of the particles during melt processing with PLGA to createsingle administration vaccine formulations. Qβ, QP-P4, and Qβ-CH401(Rat)particles were lyophilized and subjected to melt processing at 10 wt %with PLGA at 95° C. using the same method previously described. Theparticles were recovered via ethyl acetate extraction and analyzed viaFPLC, DLS, and TEM to determine the extent of particle aggregation anddenaturation in response to melt processing. Processed Qβ exhibited asmall degree of aggregation, evidenced by the appearance of a peak at 10mL in the FPLC chromatogram and at 132.3 nm in the DLS histogram (FIGS.20A-B). The total amount of Qβ aggregates from the DLS was lower thanthe result previously seen, 4.4% versus 16.2% respectively. However, theaverage size of the lower radius peak had an average value of 23.2 nmversus 12.6 nm from the previous result. This discrepancy was due tobinning effects of the lower order aggregates with the single particles,skewing the calculated amount of aggregates. The FPLC aggregate peak wasalso lower in intensity relative to the previous result and the overalldifference may have been due to slight differences in the temperatureprofile of the syringe extruder applying different levels of thermalenergy during melt processing. TEM of the processed Qβ exhibitedparticles of the correct size and morphology, verifying that theparticles maintained integrity during melt processing, consistent withprevious results (FIG. 20C).

Analysis of processed QP-P4 via FPLC and DLS indicated similar levels ofaggregation as wild type Qβ, with the appearance of a peak at 10 mL inthe chromatogram and a peak at 132.8 nm in the DLS histogram (FIGS.21A-B). TEM analysis also yielded similar result with intact particles(FIG. 21C). Processed Qβ-CH401(Rat) exhibited a small increase inaggregated species relative to wild type Qβ and had a more pronouncedaggregate peak at 10 mL on the FPLC chromatogram (FIG. 21D). The DLShistogram was in agreement with the FPLC result, and exhibited twoaggregate peaks at 129.7 and 265.2 nm (FIG. 21E). The increase inaggregation for processed Qβ-CH401(Rat) relative to Qβ-P4 particles maybe due to the CH401(Rat) peptide having more a less chargedcharacteristic. Furthermore, studies done with the chemical conjugationof these peptides to a viral nanoparticle have shown CH401(Rat)conjugates have a higher tendency to aggregate in solution than P4conjugates at higher concentrations. The factors behind the increase inaggregation for Qβ-CH401(Rat) during melt processing are unclear,however the increase is not drastic relative to wild type Qβ. Both Qβ-P4and Qβ-CH401(Rat) yielded TEM micrographs that indicated intactparticles of the correct size and shape (FIG. 21F). Overall, theincorporation of the peptide epitopes into the chimeric assemblies didnot have a deleterious effect on the physical structural properties ofthe particles after melt processing at 95° C. with PLGA.

Our previous studies with proteins and viruses melt processed with PLGAhave indicated that surface chemistry impacts the in vitro releaseprofile from the PLGA material. Thus, the in vitro release of PLGAmaterials prepared via melt processing with 10 wt % of Qβ, Qβ-P4, andQβ-CH401(Rat) was assessed to determine the effect of the surface P4 orCH401(Rat) peptide epitopes (FIG. 22.). The release study was conductedusing PBS at 37° C. to model physiological conditions for 27 days. Allsamples exhibited similar release profiles and reached a maximum of ˜56%cumulative release. The release was more rapid than 10 wt % loaded Qβmaterial studied, and this was likely due to the release buffer volumebeing half that of the previous release study. As previously discussed,in vitro release from PLGA devices is dependent on the releaseconditions. PLGA laden with Qβ-P4 did exhibit a smaller initial releaseover the first 7 days, with ˜2.5% released versus ˜7.5% for Qβ andQβ-CH401(Rat). P4 is has an overall acidic character and an isoelectricpoint of 3.3, which should increase the repulsive forces between Qβ-P4particles and the carboxylic acids present in PLGA, thereby increasingrelease. P4 may form salt bridges with lysine residues on the surface ofQβ, diminishing the acidic characteristic and increasing the hydrophobiccharacteristic of the particle which can more strongly interact with thehydrophobic portions of PLGA and slow release. CH401(Rat) has an overallslightly basic characteristic, with a pI of 9.25, and may not form theseinteractions. Nonetheless, all samples exhibited similar releaseprofiles during the bulk erosion phase after day 7. The in vitro releaseresult indicated the incorporation of peptide epitopes via geneticfusion onto Qβ melt processed with PLGA may have an effect on theinitial diffusion controlled phase of release, but it does not have asignificant effect on the release profile during the bulk erosion phase.

EXAMPLE 3

In this example, we sought to apply the melt-processing methodsdeveloped Example 2 for Qβ to create PLGA based materials laden withCPMV for intratumoral administration and extended release. In theprevious study, cylindrical implants were directly implanted into thesubcutaneous space of the mice, and direct implantation of PLGA rodsinto cancer bearing sites has previously been shown to successfullydeliver anti-cancer agents. This route of administration may not beviable for tumor sites that have limited space or accessibility;therefore, we transformed the melt processed material into a formulationthat could be injected as a suspension. Oscillatory ball milling wasused to create micron sized particles of melt processed cylindricalPLGA/CPMV material to maintain the solventless nature of the process.The PLGA/CPMV microparticles were able to be manufactured whilemaintaining CPMV particle integrity and effectively impede ovariancancer progression with a single administration. We further applied theCPMV melt processing method to create microneedle arrays to allow forthe dermal administration of CPMV as an in situ vaccine. Microneedlesallow for the facile and pain free administration of immunostimulatoryagents through application of micron sized needles on the skin.Incorporation of CPMV into PLGA microneedle arrays via melt processingwould allow for the direct administration of CPMV as an in situvaccination device to skin cancers such as melanoma. Overall, CPMV wasable to be successfully formulated into several PLGA based devices viamelt processing and maintain structural and biochemical integrity.

Materials and Methods

Poly(lactic-co-glycolic acid) (EXPANSORB® 10P019, 50:50 PLGA, inherentviscosity 0.15-0.25 dlg⁻¹, 5-20 kDa) was purchased from PCAS. Potassiumphosphate monobasic anyhdrous, potassium phosphate dibasic anydrous,sodium phosphate dibasic hetptahydrate, Gibco 1X PBS pH 7.4, butanol,Miller LB Broth, D-sucrose, sodium azide, sodium chloride, ethylacetate, 1-step PNPP substrate, PNPP tablets, Tween-20, albuminstandard, chloroform, n-butanol, carborundum, and sodium hydroxide werepurchased from Fisher Scientific. Poly(ethylene glycol) (M_(n)=8000) waspurchased from Amresco. Bradford reagent was purchased from VWR. Drymilk was purchased from LabScientific Inc. Uranyl acetate 2% solutionwas purchased from Electron Microscopy Sciences. PLGA-FPI749 waspurchased from Akina Inc. Goat anti-mouse IgG-alkaline phosphatase andgoat anti-rabbit IgG-alkaline phosphatase were purchased from LifeTechnologies. Rabbit anti-CPMV IgG and D-luciferin were a generous giftfrom Dr. Steinmetz. All reagents were used directly, without furtherpurification.

Instrumentation

Fast protein liquid chromatography (FPLC) was performed using a GEHealthcare AKTA-FPLC 900 chromatography system equipped with a Sephacryl1000 SF 10/300 size exclusion column. For all FPLC experiments, themobile phase was 50 mM phosphate buffer, with 150 mM NaCl (pH 7.4) at aflow rate of 0.4 mL/min. Samples were injected at a concentration of0.1-0.75 mg/mL and the resulting chromatograms were normalized by themaximum absorbance at 260 nm. Dynamic light scattering (DLS) experimentswere performed on a Wyatt DynaPro NanoStar DLS instrument. Samples wereanalyzed at 25° C. in plastic disposable cuvettes with a path length of10 mm. Transmission electron microscopy (TEM) was performed on a FEITechnai TF30 ST microscope. Negative stained TEM samples were mounted on400 mesh hexagonal copper grids bearing Formvar support film, stainedwith 2% uranyl acetate solution, and allowed to dry for 12 h. Microplatemeasurements were taken with a Biotek Synergy HT microplate reader.Centrifugation was performed with an Eppendorf 5424 centrifuge for spinfiltration or a Beckman Coulter Avanti J-E centrifuge for CPMVpurification. Ultracentrifugation was performed with a Beckman CoulterOptima L-100 XP ultracentrifuge. UV-vis spectra were collected using aShimadzu BioSpecNano UV-vis spectrophotometer. Scanning electronmicroscopy was performed using a JEOL-6510LV scanning electronmicroscope at 1 kV. Ball milling was performed using a FritschLaboratory Mini Grinder PULVERISETTE 23 equipped with a PTFE grindingbowl. Luminescence imaging was performed using a PerkinElmer IVISSpectrum BLI imaging system. Confocal images were collected using aLeica TCS SPE microscope with a 635 nm solid state laser.

CPMV Production and Purification

CPMV was produced and purified based on a previously published protocol.Vigna ungiuculata plants were grown for 10 days and were inoculated with50 μg of CPMV in 50 μL of pH 7, 0.01 M phosphate buffer per leaf viamechanical inoculation with a dusting of carborundum. The infection wasallowed to proceed for 10 days and the leaves exhibited extensive yellowmottling. The leaves were harvested and stored at −80 ° C. until furtherpurification.

The leaves were pulverized inside of a plastic bag by hand and then 3volumes of 4° C. phosphate buffer pH 7, 0.1 M, was added per 100 g(i.e., 300 mL per 100 g). The slurry was homogenized using a standardblender and then filtered through 3 layers of cheese cloth. The filtratewas centrifuged at 10,500 rpm for 20 minutes using with a JLA-10.500rotor. The supernatant was decanted and had 0.7 volumes of 1:1 (v/v)chloroform:n-butanol added and stirred on ice for 30 minutes. Thesolution was centrifuged at 6,000 rpm for 10 minutes using a JLA-10.500rotor. The upper aqueous phase was removed and had NaCl added to 0.2 Mconcentration and 8 kDa PEG added at 8 wt %. The mixture was stirred for30 minutes on ice and then stored at 4° C. for 2 hours. The solution wasthen centrifuged at 14,000 rpm for 15 minutes using a JLA-16.250 rotor.The supernatant was decanted and the precipitate was resuspended in 0.01M phosphate buffer, pH 7, overnight at 4° C. The solution was thencentrifuged at 9,500 rpm using a JLA-16.250 rotor and the supernatantwas collected. The supernatant was purified on 10-40% sucrose gradientsin an SW28 rotor at 28,000 rpm for 3 hours. The light scattering regionwas collected from each gradient tube and subsequently pelleted in anultracentrifuge using a 50.2Ti rotor at 42,000 rpm for 3 hours. Thepurified CPMV particles were dissolved in 0.1 M phosphate buffer, pH 7,and purity was verified via agarose gel electrophoresis, FPLC, DLS, andTEM. For melt processing, the CPMV was dialyzed into deionized water viarepeated centrifugation at 6,000 rpm using 100,000 kDa MWCO centrifugalspin filters (at least ten spins). The CPMV solution was then frozen at-20° C. and lyophilized for 72 hours. Lyophilized CPMV was resuspendedin 0.1 M phosphate buffer, pH 7, and characterized for particleintegrity via FPLC, DLS, and TEM.

CPMV Melt Processing

Poly(lactic-co-glycolic acid) (PLGA) and 8 kDa polyethylene glycol(PEG8000) were individually ground manually with a mortar and pestletwice, 10 minutes each time, into a fine powder. The PLGA powderconsisted of particles with an average length of 185.8 ±89.1 iim asdetermined via SEM image analysis. PLGA was mixed with the 10 wt % ofCPMV and 15 wt % PEG8000 via repeated vortexing in a 2 mL Eppendorftube. The custom built aluminum syringe-die utilized for melt processingof PLGA/protein blends was utilized for the melt processing ofCPMV/PLGA/PEG8000 blends. Approximately 200-350 mg of CPMV/PLGA/PEG8000blend was added into a polypropylene 1 mL volume Norm-Ject syringes andloaded into the aluminum barrel heated at 80° C. as determined by aglass thermometer for 3 minutes. The melted blend was pushed through the1 mm circular die manually and the resulting cylindrical implants haddiameters ranging from 1.0-1.1 mm. For fluorescent material, 5 wt % ofPLGA tagged with FPI749 fluorescent die was added to the blend mixtureprior to vortexting and melt processed using the same method.

CPMV Recovery or Release and Characterization

Rapid CPMV recovery from implants was performed by dissolving -50 mg ofmaterial in 2 mL of ethyl acetate for 15 minutes. The solution wascentrifuged for 5 minutes at 5,000 rpm using an Eppendorf 5424centrifuge with a fixed angle rotor. The supernatant was decanted andthe process was repeated two more times. The remaining solids were driedunder vacuum at room temperature for 24 hours. The solid proteinrecovered was resuspended in 0.01 M phosphate buffer, pH 7, for 24 hoursat 4° C. In order to remove free RNA, the resuspended samples werefiltered using 10 kDa MWCO centrifugal spin filters for at least 10filtrations. For released samples, ˜50 mg of material was incubated in250 μL of 0.01 M phosphate buffer, pH 7, at 37° C. and the buffer wasremoved after 24 hours. The samples were filtered using 10 kDa MWCOcentrifugal spin filters for at least 10 filtrations. All samples wereanalyzed for particle integrity and RNA packaging via agarose gelelectrophoresis, FPLC, DLS, and TEM.

CPMV/PLGA/PEG8000 Microparticle Production

Prior to ball milling, 100-150 mg of cylindrical CPMV/PLGA/PEG8000material was incubated at −80° C. for 1 hour. The cylinders, in ˜2 cmlengths, were added to the PTFE grinding bowl with one 10 mm stainlesssteel grinding ball and the bowl was filled with liquid nitrogen. Thematerial was then milled at 30 Hz for 15 minutes and recovered. Theresulting microparticles were imaged via SEM and the diameters of theparticles was measured via ImageJ. The diameter distribution wasdetermined from 152 measurements of particles in two images andconverted to a frequency plot with a bin size of 2 Confocal images ofmicoparticles created with material containing 5 wt % PLGA-FPI749 wereacquired using an excitation wavelength of 635 nm and the emission wasmeasured from 700-800 nm.

Release Properties of Melt Processed CPMV/PLGA/PEG8000

Release studies were conducted on samples of the melt processed implants(-1 cm long, 9-13 mg, n=3) or microparticles (10-11 mg, n=3). Sampleswere placed in 2 mL Eppendorf tubes with 200 μL of Gibco 1X PBS with0.01 wt % sodium azide and incubated at 37° C. with 90% relativehumidity. Aliquots of 175 μL were removed at each time point andreplaced with fresh buffer. The settled microparticles were notdisturbed during the removal and replenishment of buffer. The CPMVconcentration at each time point was determined via Bradford assay withcomparison to a freshly prepared bovine serum albumin standard curve.The UV-vis extinction coefficient was not utilized for quantificationdue to the presence of empty CPMV and free RNA that would skew theUV-vis absorbance.

Microneedle Fabrication and Analysis

Microneedle arrays were fabricated utilizing either PLGA/PEG8000 orCPMV/PLGA/PEG8000 cylinders that were initially melt processed using thesyringe die extrusion device at 80° C. for 3 minutes. -2 cm lengths ofmaterial were loaded into silicone microneedle molds (MicropointTechnologies) and incubated in a vacuum oven at 80° C. The siliconemolds were designed to yield an array of 10×10 needles with 100×100 μmlength base and 250 μm height with a pyramidal shape. The samples weresubjected to vacuum for 4 minutes to remove air bubbles from the meltedmaterial and then vented to atmospheric pressure for 4 minutes to allowfor the material to fill the mold. This process was repeated 2 moretimes for a total process time of 24 minutes. The filled molds were thenmoved to −20° C. for 30 minutes and removed from the molds.

The resulting microneedle arrays of PLGA/PEG8000 were analyzed forneedle morphology via SEM. The arrays were not sputter coated prior toSEM imaging, due to needle degradation during the sputter coatingprocess. The mechanical properties of the needles were measured viacompression testing with a rate of 10 μm/s. The maximum strength of theneedle was determined from the force value at saturation. The integrityof CPMV after microneedle molding was determined from particlesrecovered via ethyl acetate extraction as previously described. Therecovered CPMV was analyzed via agarose gel electrophoresis, DLS, andTEM.

ELISA Analysis of Processed CPMV

ELISA was utilized in order to determine the retention of thebiochemical surface characteristic of CPMV after melt processing andmicroneedle molding. The melt processed and microneedle molded sampleswere spin filtered as previously described and the concentration wasdetermined via Bradford assay. Nunc Maxisorp 96-well plates with 1 μg ofCPMV sample in 200 μL of PBS, pH 7.4 at 4° C. overnight. The wells werethen blocked with 200 μL of blocking buffer (2.5% w/v dry milk in PBS,pH 7.4) at 37° C. for 1 hour. The wells were then incubated with 2 μg/mLof polyclonal rabbit anti-CPMV IgG in 100 μL blocking buffer for 2 hoursat 37° C. The wells were then incubated with 100 μL of a 0.6 μg/mL inblocking buffer of alkaline-phosphatase labeled goat anti-rabbit IgG for1 hour at 37° C. The wells were washed between each incubation stepusing 3×200 μL of 0.1% w/v Tween-20 in PBS, pH 7.4. The wells weredeveloped using 100 μL of 1-Step PNPP substrate at 4° C. for 10 minutes.The reaction was stopped with 100 μL of 2 M NaOH and the absorbance wasread at 405 nm in triplicate for each sample. The averages and standarddeviations were normalized to the wild type CPMV response to yield apercent response. Values are expressed as the average and standarddeviation of 2 measurements relative to the wild type CPMV result.

OVCA In Vivo Study

All experiments were carried out in accordance with Case Western ReserveUniversity's Institutional Animal Care and Use Committee.Hyperaggressive ovarian ID8-Defb291Vegf cancer cells (a generous giftfrom Dr. Steven Fiering, Geisel School of Medicine, Dartmouth College)were transformed to express luciferase and grown following a previouslyestablished protocol. Female C57BL/6J mice (Charles River) aged 6 weeks(n=5 per treatment group) were injected with 10⁶ luciferin positiveID8-Debf29/Vegf-A ovarian cancer cells in the intraperitoneal spaceusing a 29G insulin syringe. On day 7, two groups of 5 mice each wereinjected with either 12 mg of PLGA/PEG8000 microparticles or 10 wt %CPMV loaded PLGA/PEG8000 microparticles. The microparticles weresuspended in 1 mL of sterile PBS, pH 7.4, and immediately injected intothe intraperitoneal space utilizing a 22.5G syringe. The mass ofinjected material was chosen to deliver 300 μg of CPMV over 20 daysbased on the in vitro release profile, correlating to 3 injections of100 μg CPMV on a weekly basis for standard treatment. The othertreatment groups injected with 30, 100, and 500 μg doses of CPMV 4 timeson a weekly basis starting on day 7. The mice were studied for tumorgrowth via tumor luminescence, abdominal circumference, and weight on abiweekly basis starting on day 11. The luminescence was determined byinjection of 150 μL of luciferin into the intraperitoneal space andincubation for 5 minutes. The luminescence was then measured for 3minutes and the total photons were calculated from the luminescenceimage. The results for total luminescence, circumference, and mass werereported as the average and standard deviation of 5 mice.

ELISA was utilized to determine the level of anti-CPMV IgG generation asa result of CPMV loaded microparticle administration, blank PLGA/PEG8000microparticles, and weekly 100 μg CPMV injections. Retro-orbital bleedswere conducted on 2 mice from each group on day 46 utilizing heparinizedcapillary tubes and -100 μL of blood was collected in VACUETTE™MiniCollect™ tubes. Serum was separated by centrifuging blood samples at14,800 rpm, 4° C., for 10 min and stored at 4° C. until analyzed viaenzyme-linked immunosorbent-assay (ELISA). The anti-CPMV IgG responsewas measured by first coating Nunc Maxisorp 96-well plates with 1 μg ofCPMV in 200 μL of sterile PBS, pH 7.4 at 4° C. overnight. The wells werethen blocked with 200 μL of blocking buffer (2.5% w/v dry milk in PBS,pH 7.4) at 37° C. for 1 hour. The wells were then incubated with mousesera at 1:500, 1:2500, and 1:12500 dilutions in 100 μL blocking bufferfor 2 hours at 37° C. The wells were then incubated with 100 μL of a1:1000 dilution in blocking buffer of alkaline-phosphatase labeled goatanti-mouse IgG for 1 hour at 37° C. The wells were washed between eachincubation step using 3X 200 μL of 0.1% w/v Tween-20 in PBS, pH 7.4. Thewells were developed using 100 μL of PNPP substrate at 4° C. for 10minutes. The reaction was stopped with 100 μL of 2 M NaOH and theabsorbance was read at 405 nm in triplicate for each sample. Values wereexpressed as the average and standard deviation of 2 mice.

Results CPMV Melt-Processing

Vigna unguiculata plants were infected with previously purified CPMV andthe infection was allowed to progress until the leaves had extensiveyellow mottling. CPMV was purified from the infected leaves andcharacterized via FPLC, DLS, and TEM in a similar manner to Qβ (FIGS.23A-C). The FPLC chromatogram yielded a single peak with no aggregatesand the DLS also had a single population with an average radius of 15.2nm, in good agreement with the known size of CPMV. TEM images alsoindicated icosahedral particles of the correct size, indicating thatCPMV was successfully isolated and purified. The purified CPMV was thenspin-filtered into deionized water and lyophilized to yield a slightlyyellow powder. Resuspension of the lyophilized CPMV with 0.01 Mphosphate buffer and analysis via FPLC, DLS, and TEM did not indicateany particle disassociation or aggregation as a result of thelyophilization process.

Following the lyophilization, CPMV was subjected to melt processing withPLGA in a similar manner to Qβ. A blend of 10 wt % CPMV and PLGA wasmanually mixed via repeated vortexing and heated at 95° C. for 10minutes using the syringe extrusion device to create CPMV laden PLGAcylinders. Intact CPMV could not be recovered from these samples and allFPLC, DLS, and TEM results indicated that the melt processing conditionsused for Qβ previously resulted in complete denaturation of CPMV. Thisis likely due to the Qβ coat proteins being interconnected via covalentdisulfide linkages, while CPMV coat proteins are stabilized throughnon-covalent interactions potentially making it more sensitive toapplied thermal energy. To prevent particle denaturation, both theapplied temperature and incubation time of the blend was lowered to 80°C. and 3 minutes respectively. 100% PLGA was unable to be extruded underthese conditions, due to the polymer not fully reaching the melt stateand being too viscous to push through the extrusion die. 8 kDa PEG(PEG8000) was added at 15 wt % to act as a plasticizer and bring themelt viscosity down to a level that can be extruded under theseconditions. Furthermore, PEG additives in protein-PLGA systems hasprevented some aggregation with Qβ in our studies and has been shown toenhance protein stability in microsphere formulations. Therefore, theaddition of PEG may also enhance the stability of CPMV in the melt.

CPMV was melt processed at 10 wt % with the PLGA/15%PEG8000 blend andwas able to be recovered successfully from the extruded polymer sample.FPLC analysis of extracted CPMV yielded two, overlapping Gaussian peaksforming a single skewed peak. These two peaks, with maxima at 18.1 mLand 21.1 mL, corresponded to CPMV particles and disassociated RNArespectively. Spin filtering the CPMV and free RNA sample to remove freeRNA yielded an FPLC chromatogram, consisting of only the CPMV particlepeak (FIG. 23A). The spin-filter used had a 10 kDa MWCO, smaller thanfree CPMV coat proteins (41.2 and 23.7 kDa for the L and S subunitproteins respectively), to ensure only free RNA was removed and not anypotentially disassociated coat proteins. The spin filtration did removethe RNA, however the full length RNA1 and RNA2 associated with CPMV are5.8 and 3.4 kilobase in length respectively. These lengths of RNA wouldexhibit hydrodynamic radii in the range of 9-12 nm when free insolution, suggesting the full length RNA would not be removed using the10 kDa MWCO filter which has an approximate pore size of 10 A in radius.The successful removal of the RNA using the spin filters suggests thatthe RNA is released and degraded as a result of melt processing. RNA issensitive to high temperatures and shear, which are applied during themelt processing, thus the degradation of RNA under these conditions waslikely. An aggregate peak was also observed at the void volume, howeverit was not the majority of the eluted particles. The peak did alsobroaden towards higher elution volumes after melt processing, possiblyindicating some lower order aggregates small enough to not elute in thevoid volume. The ratio of 260 to 280 nm absorbance is 1.8 for wild typeCPMV and exhibits a maximum absorbance at 260 nm due to the contributionof the packaged RNA. After removal of the RNA, the empty CPMV particlesexhibit a maximum absorbance at 280 nm and a 260 to 280 nm ratio of 0.9.The FPLC result of the recovered melt processed CPMV particles and theshift in 260 and 280 nm observed was consistent with the knownspectroscopic properties of CPMV devoid of RNA. The DLS result matchedwell with the FPLC result, and yielded two peaks at 16.9 and 147.9 nm(FIG. 23B). The peak at 147.9 nm likely correlated to the aggregateseluting in the void volume, while the increase in the lower valueaverage radius from 15.1 to 16.9 nm would correlate to the broadeningseen in the FPLC result. TEM imaging further confirmed that theparticles remain intact and in the correct shape after melt processingwith the PLGA/PEG8000 blend.

Agarose gel electrophoresis was used to probe whether the meltprocessing or the organic extraction of the CPMV from the polymer causesthe RNA to disassociate from CPMV. Three samples of 10%CPMV/PLGA/PEG8000were incubated in 10 mM phosphate buffer, pH 7.4 at 37 ° C. and thereleased CPMV after 24 hours was collected, pooled, and concentrated.The organic extracted CPMV, released CPMV, and wild type CPMV were runon an agarose gel with 1 wt % ethidium bromide and imaged via UV for RNAvisualization, and Coomassie staining for protein visualization (FIG.24). The wild type CPMV exhibited the expected two bands or RNA thatco-localized with the protein bands. CPMV recovered via organicextraction was representative of all of the CPMV present in the polymerand had strong signal from the RNA below the protein bands, indicatingthe RNA was disassociated from the virus particles. There was also someprotein and RNA content that remained in the well, corresponding toaggregated species and also indicating that the aggregates retain RNAwhen they form. The CPMV released over 24 hours did not have any RNAsignal, but did have the expected protein bands from intact particles.The absence of RNA from the aqueous release was likely due to theremoval of RNA during centrifugal filtration and concentration. Theresult did indicate that the organic extraction process did not causethe disassociation of RNA from CPMV, since CPMV recovered via aqueousrelease did not have RNA associated with the particles. Overall, theresults clearly indicated that CPMV could be melt processed at 80° C.with a blend of PLGA/PEG8000 and remain as intact viral nanoparticles,but with the loss of the packaged RNA.

CPMV/PLGA Microparticles

The cylindrical polymeric implants containing 10 wt % Qβ were directlyimplanted into the subcutaneous space of the mice for vaccinationagainst the Qβ surface epitopes. The utilization of CPMV as an in situvaccine requires the CPMV to be released directly into the intratumoralspace, which may not be amenable to the implantation of a cylindrical orother geometry solid polymeric piece. Microparticles have beenextensively used as injectable polymeric depots for sustained deliveryapplications, however as discussed in the background they are almostexclusively produced through solvent based emulsion methods. In order tomaintain a solvent-less, high throughput production method oscillatoryball milling was utilized to mill the extruded CPMV/polymer cylinderinto micron sized particles. The CPMV/polymer cylinders were kept at−80° C. for 2 hours and then milled in a Teflon grinding bowl, withliquid nitrogen added, for 15 minutes at 30 Hz. The pre-freezing andaddition of liquid nitrogen during milling were necessary to maintainthe rigidity of the material so it would be effectively ground, and toprevent heating due to kinetic energy, which results in the melting ofthe polymeric material during milling.

Micron sized particles of PLGA/15%PEG8000 containing 10 wt % CPMV weresuccessfully manufactured through the cryo-milling process and exhibitedmostly elliptical morphology with rough surface characteristic whenanalyzed via SEM (FIG. 25A). Analysis of the particles with ImageJindicated that the particles predominately ranged from 10 to 20 μm indiameter (FIG. 25B), a size that has previously been used as injectabledepots for drug delivery. The microparticle size was also analyzed viaDLS after suspension in phosphate buffered saline and the suspendedparticles exhibited a similar size range to the size range determinedvia SEM (FIG. 25C). The bimodal characteristic of the DLS histogram isnot reflected in the image analysis of the SEM image, and may be aconsequence of microparticle settling during DLS measurement skewing theresult. Fluorescently tagged PLGA was also incorporated with theCPMV/polymeric material in order to allow for the study of PLGAdegradation once injected in vivo. PLGA was purchased coupled with anFPI749 near-infrared dye, useful in biological tissues as the excitationand emission of NIR dyes allow for good penetration of tissues and lowauto-fluorescence background signal. PLGA-FPI749 was incorporated at 5wt % with the CPMV/PLGA/PEG8000 blend via melt processing as previouslydescribed. The extruded material was then cryo-milled intomicroparticles and imaged via confocal microscopy (FIG. 25D). Themicroparticles were fluorescent under confocal imaging, indicating thatthe dye-labeled PLGA maintained fluorescence during the melt processingat 80° C. and can potentially be used to monitor in vivo PLGAdegradation.

In order to ensure that CPMV did not denature or further aggregateduring cryo-milling to create the microparticles, CPMV was recoveredfrom the microparticles via release into phosphate buffered saline over24hours. Analysis of the recovered CPMV on an agarose gel indicatedsimilar results to CPMV only subjected to melt processing (FIG. 26A).The CPMV from microparticles did not retain the RNA and was maintainedas a viral nanoparticle. This was further confirmed via DLS analysis,and the CPMV did not exhibit increased aggregation after cryo-millinginto microparticles relative to initial melt processing (FIG. 26B).

In Vitro CPMV Release

The in vitro release properties were studied to determine how CPMV wouldrelease from the polymeric matrix and the effect of material geometry(FIG. 27). Rod shaped samples produced via initial melt processing withthe syringe extrusion device were compared to polymeric microparticles.Both samples were incubated with phosphate buffered saline (pH 7.4) at37° C. to simulate in vivo conditions. The burst release from theinitial hydration and swelling of the polymeric materials resulted in 10and 5% cumulative protein release for rod and microparticle samplesrespectively. After the initial hydration, both samples followedrelatively linear release profiles over a 40 day period. PLGA devicestypically follow a biphasic release profile, with a lag period afterinitial hydration until bulk erosion begins. The addition of the PEGadditive has previously mitigated the lag effect in PLGA samples withQβ. The PEG8000 additive with the CPMV/PLGA samples resulted inincreased porosity of the PLGA matrix after the initial hydration, dueto the hydrophilic PEG diffusing out of the matrix. These pores wouldallow for more rapid diffusion of CPMV out of the matrix and the morerapid onset of bulk erosion during release. After 40 days, the totalcumulative release of CPMV was -40 and -25% for the rod andmicroparticle samples respectively.

The lower total amount released for the microparticle samples wasunexpected, as the increased surface area of the microparticles relativeto the rod shaped samples should result in a higher total amountreleased. This discrepancy is likely due to how the different geometriesrespond to swelling at 37° C., above the glass transition temperature ofPLGA. The rod geometry of the initial melt processed material remains asa rod once the swelling and softening of the material takes place duringrelease and does not collapse into itself to form a circular geometry.In contrast, the microparticles settle to the bottom of the vesselcontaining the material and release medium. Once the swelling andsoftening of the material takes place, the particles coalesce into asingle piece of polymeric material, limiting the available surface areadirectly in contact with the release medium. This difference between therod shaped and microparticle samples results in the rod geometry havinga larger surface area relative to the coalesced microparticle samples,thus likely being the reason for the rod shaped initial melt processedgeometry having a larger total amount of protein released over time.This effect for microparticle samples during in vitro release studieshas been studied previously, and the effects can be mitigated throughvortexing to break-up the coalesced particles, sonication and sieving,and other methods to prevent the consolidation of particles into asingle polymeric material with limited release surface area. All ofthese methods would potentially alter the release profile towards higheramounts released, however each skews the release kinetics throughapplication of mechanical and mixing forces that can break-up polymerentanglements and alter the diffusion of proteins out of the polymericmatrix. These is no consistent method for determining completelyaccurate in vitro release profiles and in vivo application would exhibitdifferent release profiles than in vitro due to the microparticles beinginjected into viscous tissue, a constant flux of water through theenvironment, and enzymatic activity on the polymer. Overall, the releaseprofile allowed for a rough estimation of the release profile for invivo application and demonstrates that, in both initial melt processedand microparticle formulations, CPMV could be successfully released andmaintain particle integrity.

The CPMV was not completely released from the polymer matrix over thecourse of the in vitro release study for both rod shaped andmicroparticle CPMV/polymer samples. The maximum cumulative amountreleased was 42% and 27.3% for rod and microparticle samplesrespectively. These values are both lower than the release values seenwith Qβ processed with 10% PEG8000, which was 72% at day 45. Thisdiscrepancy between released amounts was surprising, as an increase inPEG additive was expected to increase the release rate due to increasedporosity as the PEG diffuses out. However, PEG also functions as aviscosity modifier when present in high concentrations in solution.Studies with PEG8000 have found that 10 and 20 wt % solutions of PEG8000exhibit viscosities 9 and 20 times higher than water respectively.Furthermore, PEG8000 acts as a precipitating agent for many proteins andit has previously been shown that PEG polymers above 6000 Da atconcentrations above 15 wt % in solution are highly effective atprecipitating high molecular weight proteins. Thus, in the in vitrorelease environment the PEG may remain entrapped in the PLGA with theCPMV and increase the local viscosity within the channels formed in thepolymer matrix during swelling and pore formation. This would retard thediffusion of both PEG and CPMV through the matrix into the externalenvironment, diminishing the effectiveness of PEG as a porogen when usedas at higher concentrations. The increased local concentration ofdissolved PEG within water channels can also potentially result in thelocal precipitation of CPMV within PLGA, further slowing the releasefrom the matrix. Local CPMV precipitates will solubilize as intact whenthe PEG concentration in the solution decreases, and PEG precipitationis a step in the purification of the virus from infected leaves.However, the diffusion of PEG from the polymer matrix will be controlledby the volume of the in vitro release buffer and sampling times. Therelease in the in vitro system is not wholly representative of in vivorelease for reasons previously described, and the proposed issues withPEG diffusion and CPMV precipitation will likely be mitigated byconstant release medium flux in vivo. The difference between in vitroand in vivo release and degradation of the polymer has previously beenobserved, where implanted Qβ/PLGA systems were completely degraded after75 days and in vitro release systems of the same material still hadunreleased Qβ and polymeric material present after the same time period.The microparticle systems for CPMV would have the same limitations dueto the PEG additive as the rod samples, but the effect of high local PEGconcentration would be enhanced due to the coalescence of the particlesas previously described. This would further diminish the diffusion ofPEG out of the polymer matrix and result in the low amount of releaseexhibited during the in vitro release study.

Microneedle Production

The composite CPMV/PLGA/PEG8000 material application was furtherexpanded to microneedle arrays produced via melt processing. Aspreviously described in Example 2, microneedles are an attractiveplatform for the non-invasive and painless administration of vaccinesand Qβ was shown to be successfully incorporated into microneedlearrays. The application for Qβ was to generate humoral immunity againstQβ carrying an antigenic epitope on the surface. For CPMV, theapplication is in situ vaccination where the viral nanoparticle isdirectly applied to the tumor site. This limits the application of CPMVmicroneedle arrays to melanoma treatments, however melanoma is a commonand serious cancer that can spread to other parts of the body andmetastasize. Administration of CPMV for immunotherapy against melanomavia microneedle application has the potential to treat melanoma, andalso protect against metastasis. Studies with CPMV have shown that insitu vaccination generated a humoral immune response against the cancercells and protected against tumor growth when the animal model wasre-challenged at a different site.

Microneedle arrays were successfully manufactured utilizing the samesilicone molds that were previously utilized to create microneedles withQβ. PLGA with 15 wt % PEG8000 and no CPMV was first utilized to makeseveral microneedle arrays to ensure the incorporation of PEG did notadversely affect the mechanical properties of the needles. The needleswere uniform and of the correct size via SEM after melt molding at 80°C. with 3 degassing cycles for 25 minutes total processing time (FIG.28A). The mechanical properties were determined via compression testingand yielded values of 0.376 ±0.00835 N maximum strength per needle and37.55 ±0.835 N maximum strength for the entire array (FIG. 28B). Themaximum strength values were determined from 3 individual arrays,indicating that the melt molding process results is reproducible andconsistent in the strength of the needles produced. These values havepreviously been shown to be sufficient for both in vivo and clinicalmodels for skin puncture by the microneedles. This result demonstratedthat the PLGA/PEG8000 blend can be successfully molded into microneedlesand exhibit the necessary mechanical properties for application.

After validation of microneedle arrays production using PLGA/PEG8000,the melt processed CPMV/polymeric material was melt molded intomicroneedle arrays using the same conditions for PLGA/PEG8000microneedle production. The arrays formed exhibited the same needleshape and size and the CPMV within the array was extracted via ethylacetate as previously described. The recovered CPMV was analyzed foraggregation and particle integrity. The DLS histogram of the recoveredparticles indicated 51.2% of the particles remained as intact, singleparticles (FIG. 29A). The remaining particles appeared as two aggregatedpopulations centered at 126.7 and 235.2 nm roughly correlating toaggregates of 8 and 15 particles respectively. Analysis of the particleswith agarose gel electrophoresis further confirmed that the particlesremained intact, but lost the viral RNA packaged with CPMV (FIG. 29B).The extracted particles were also analyzed via TEM, and the images didhave intact particles present (FIG. 29C). However, there was also strongsignal from polymer still present in the sample obscuring some of theparticles. This indicated that the extraction of the particles from themicroneedle polymeric array was did not successfully remove all of thepolymer. The mass of polymer composite used, volume of ethyl acetateused, and overall process were the same as previous extraction methodsthat successfully separated protein from the polymer matrix. Thisincomplete extraction may be due to the different geometry of themicroneedle array, square as opposed to the typical rod geometry,hindering effective solubilization of all of the polymer in the sample.The presence of free PLGA in solution may have also skewed the DLSresult, as free PLGA could potentially interact with multiple virusesthrough ionic or hydrogen bonding interactions, causing them toaggregate in solution. Nonetheless, the results indicated that furthermelt molding into microneedle arrays kept the majority of CPMV intactand have potential application for dermal administration of CPMV as anin situ vaccination agent for melanoma and other skin cancers.

The previous work described herein has demonstrated that CPMV can bemelt processed and retain structural integrity with minimal to moderateaggregation depending on the time of the melt processing. However, allof the analytical techniques utilized were only able to evaluate thestructural integrity, size, and association between the coat proteinassembly and viral RNA of the CPMV. ELISA was utilized to determine howthe biochemical signature of CPMV was maintained after melt processingwith the PLGA/PEG8000 blend. The maintenance of the biochemical patternson the surface of CPMV is essential for the successful application as anin situ vaccine, as the PAMPs on the surface are necessary to elicit astrong anti-tumoral response. The ELISA study utilized a polyclonalantibody purified from rabbit specifically against CPMV thatspecifically recognize the pattern of the coat protein assembly on theviral surface. ELISA plates were coated with wild type CPMV, CPMVextracted from rod samples that were melt processed with the syringeextruder, and CPMV extracted from the further melt processed microneedlearray. The CPMV from the melt processed samples was extracted via ethylacetate to represent all of the CPMV present in the sample. The meltprocessed viruses were also spin filtered to remove all free RNA afterextracting to ensure the ELISA response would only be due to the coatprotein assemble of CPMV. The results of the ELISA analysis werenormalized to the response from wild type CPMV (FIG. 30). CPMV subjectedto the initial melt processing exhibited an antibody recognition of 93.2±2.1% of the wild type CPMV recognition. Analysis via a two tailedt-test against wild type CPMV indicated that there was no statisticaldifference between the ELISA response of wild type and melt processedCPMV. Therefore, within error, the melt processing via syringe extrusionhad no effect on the biochemical signature of CPMV. Further meltprocessing to create microneedle arrays yielded CPMV with an antibodyrecognition of 92.4 ±5.3% relative to the wild type. Statisticalanalysis of the microneedle processed CPMV against wild type CPMVyielded a p value less than 0.05, indicating that there was statisticaldifference between the samples. The lowered recognition due to meltprocessing could potentially be due to some denaturation of the particleor decreased surface for antibody binding due to aggregation jammingparticles together. However, the diminishment in antibody recognition ofthe microneedle sample was only 7.6% indicating that the majority ofparticles retain the biochemical character of CPMV after extensive meltprocessing.

Ovarian Cancer Treatment with CPMV/PLGA/PEG8000 Microparticles

CPMV has previously been shown to effectively treat anID8-Debfb29/Vegf-A aggressive ovarian cancer (OVCA) model in a mice whenadministered in weekly doses. We sought to utilize CPMV formulated intothe polymer blend via melt processing and milling into microparticles asa single administration depot to replace the multiple injections of CPMVnecessary for treatment. As the melt processed formulation has beenshown to maintain the structural and biochemical properties of wild typeCPMV and the ability to be released, we hypothesized that a singleinjection of microparticles into the intraperitoneal space of the micecan successfully release CPMV and elicit an anti-tumoral response. Micewere inoculated via intraperitoneal injection with the aggressiveovarian cancer ID8-Debfb29/Vegf-A cells transformed to expressluciferase as a reporter for tumor growth. Treatment began 7 days aftertumor cell inoculation with 12 mg of CPMV microparticles suspended in 1mL of PBS and injected into the intraperitoneal space of each treatedmouse. This amount of microparticles was chosen to release 300 μg over20 days based on the in vitro release study and would correlate to 3weekly injections of CPMV in 100 μg doses. The control group wereinjected with the same amount of PLGA/PEG8000 microparticles withoutCPMV present. Three other treatment groups were injected 4 times on aweekly basis with 30, 100, or 500 μg of CPMV in solution (FIG. 31A).

Tumor growth was monitored on a biweekly basis by measuring theluminescence from the luciferase reporter in the OVCA cells, thecircumference of the mouse abdomen, and the weight (FIGS. 31B-C). Theluminescence result both indicated that the single administration ofPLGA/PEG8000 microparticles loaded with CPMV had a moderate effect inpreventing tumor growth, with the luminescence value of mice treatedwith the microparticles having roughly 50% the luminescence of thecontrol group at day 57. The mice treated with multiple injections ofCPMV at all dosages exhibited higher suppression of tumor growthrelative to the microparticle and control group, exhibited totalluminescence values 75% lower than the control group. The abdominalcircumference measurement was also indicative of OVCA tumor growth dueto the fluid retention and swelling in response to the spread of OVCA.Circumference measurements yielded similar results to the luminescencemeasurements, with CPMV loaded microparticle treated mice having anincrease in circumference lower than the control group but higher thanthe mice treated via repeat injection. The weight measurements also hada lower average weight for the CPMV microparticle treatment group andthe control group, while the CPMV injection treated group wasconsistently lower than both groups. Luminescence images of thetreatment groups on day 57 (FIG. 32) clearly showed the CPMVmicroparticle group had lower tumor growth than the control group.

Effective stimulation of the immune system against CPMV is essential forthe simultaneous generation of an immune response against the OVCA tumorantigens for anti-tumoral activity. We had previously shown that theCPMV subjected to melt processed retained recognition by anti-CPMV IgGand sought to demonstrate that treatment with the CPMV loadedmicroparticles generated similar levels of anti-CPMV IgG as the micetreated with repeated injections of a CPMV solution. Mice that weretreated with the PLGA/PEG8000 microparticles, the CPMV loadedmicroparticles, and repeated 100 μg injections had sera collected viaretro-orbital bleeds on day 46 from 2 mice for each group to determinethe anti-CPMV IgG levels. The sera was assayed via ELISA against wildtype CPMV for 3 different dilution levels to ensure signal saturationwas not occurring from the ELISA result (FIG. 33). The mice treated with100 μg CPMV and the CPMV microparticles exhibited similar levels ofanti-CPMV IgG for all dilutions, while the control microparticle micehad no response as expected. This result demonstrated that the CPMVmicroparticles release intact CPMV in vivo and elicit a similar immuneresponse as repeated injections of CPMV, validating that the CPMVpolymeric devices can release CPMV over an extended period of time invivo and serve as a single administration vaccine.

Ovarian Cancer Treatment with CPMV/PLGA/PEG8000 MicroparticlesCo-Administered with Soluble CPGMV

The results in the in vivo OVCA treatment example demonstrated that theCPMV microparticles suppressed tumor growth in an aggressive ovariancancer model relative to untreated group. However, the CPMV particleswere not as efficacious as repeated administration of CPMV solution. Inthe present example, we sought to overcome these effects by theco-administration of CPMV in solution alongside the microparticles toprovide an initial immune response before sustained CPMV release. Micewere inoculated via intraperitoneal injection with the aggressiveovarian cancer ID8-Debfb29/Vegf-A cells transformed to expressluciferase as a reporter for tumor growth. Treatment began 7 days aftertumor cell inoculation with CPMV microparticles suspended and solubleCPMV in PBS and injected into the intraperitoneal space of each treatedmouse. The control group was injected with the same amount ofPLGA/PEG8000 microparticles without CPMV present and with CPMV insolution.

Tumor growth was monitored on a biweekly basis by measuring theluminescence from the luciferase reporter in the OVCA cells (FIGS. 34).The luminescence result indicated that the single administration ofPLGA/PEG8000 microparticles loaded with CPMV and with CPMV in solutionexhibited enhanced suppression of tumor growth, with exhibited totalluminescence values 75% lower than the control group at day 57.

EXAMPLE 4

This example describes the development of a single-dose HPV vaccine inwhich L2 peptide antigens from the high-risk HPV16 strain were displayedon the surface of (virus-like particles) VLPs derived from bacteriophageQβ, a carrier used in several other vaccine candidates. We chose abioconjugate chemistry protocol to achieve multivalent presentation ofL2 antigens on the Qβ carrier. A combination of size exclusionchromatography (SEC), transmission electron microscopy (TEM), anddynamic light scattering (DLS), and gel electrophoresis was performed tovalidate the structural integrity and epitope display of the HPV-Qβ.Immunogenicity was validated in healthy mice and the antibody titers andsubtypes were determined using the ELISA-based protocols. The HPV-Qβparticles were then encapsulated in poly(lactic-co-glycolic acid) (PLGA)implants, using a benchtop melt-processing system, and implanted intomice. We monitored the release rate of the VLPs in vitro and in vivo andconfirmed that the HPV-Qβ particles released remained intact. Finally,immunizations were carried out by comparing the implant vs. solubleinjections. Data showed that the antibody titers generated by thesingle-dose HPV-QP/PLGA implant were equivalent to traditional injectionschedules and played a neutralizing role against the HPV pseudovirus.

Materials and Methods Expression of Qβ

Bacteriophage Qβ VLPs were expressed and purified, as previouslydescribed. Chemically competent BL21(DE3) Escherichia coli cells (NewEngland Biolabs) transformed with pET28CP containing the Qβ coat proteinsequence were plated onto lysogeny broth (LB) agar (Thermo FisherScientific) containing 50₁.tg/mL kanamycin (Gold Biotechnology). Theisolated colonies were picked into 100 mL of autoclaved LB medium pluskanamycin and cultured for 12 h at 37° C. to saturation. We added 50 mLof the culture to 1 L MagicMedia (Thermo Fisher Scientific, Carlsbad,Calif., USA) and incubated at 37° C. for another 24 h, shaking at 300rpm. The cell pellets were collected by centrifugation at 1500×g andfrozen at −80 ° C. overnight. The pelleted cells were resuspended in 100mL phosphate-buffered saline (PBS) on ice and lysed using a probesonicator for 10 min. The lysate was centrifuged at 10,080×g, thesupernatant was collected, and the Qβ particles were precipitated byadding 10% w/v PEG8000 (Thermo Fisher Scientific) at 4° C. for 12 h on arotisserie. The precipitated fraction was pelleted by centrifugation at10,080x g and dissolved in 40 mL PBS before extraction with a 1:1 v/vbutanol/chloroform. The aqueous fraction was collected by centrifugationas above (at 10,080×g) and Qβ particles were purified on a 10-40%sucrose velocity gradient by ultracentrifugation at 96,281×g for 4.5 h.The light-scattering Qβ layer was collected and pelleted byultracentrifugation at 160,326×g for 3 h. The purified Qβ particles weredissolved in PBS as a stock solution for later experiments.

Synthesis of HPV-Qβ

HPV peptides were conjugated to Qβ via an SM(PEG)8 bifunctional linkercontaining an NHS group and a maleimide group (Thermo FisherScientific). In brief, 20 mg of Qβ was mixed with a 500-fold molarexcess of SM(PEG)8 to Qβ in 0.5 mL PBS (pH 7.4), at room temperature for1 h. The unreacted SM(PEG)8 was removed using a 100 kDa cut-offcentrifuge filter at 3000×g for 15 min, and the recovered VLPs werewashed with 0.5 mL PBS. The washed VLPs were resuspended in 0.5 mL PBSand reacted with a 500-fold molar excess of HPV L2 peptides at roomtemperature for 2 h. The unreacted peptides were removed by centrifugalfiltration as above; the recovered HPV-Qβ vaccine candidates were washedtwice in 0.5 mL deionized water, and then dialyzed against deionizedwater for 24 h.

Synthesis of Qβ-Cy5

Bacteriophage Qβ (20 mg) was labeled with Cy5 through conjugation via a500-fold molar excess of sulfo-Cy5 NHS ester (Lumiprobe) in 0.5 mL 0.1 Mpotassium phosphate buffer (referred to as KP buffer; K₂HPO₄ and KH₂PO₄,pH 8.3), for 4 h at room temperature. The unreacted components wereremoved by centrifugal filtration as above, and the Cy5-Qβ particleswere dialyzed against and then stored in deionized water.

Preparation of Qβ-Loaded PLGA Implants through Melt-Extrusion

The loaded PLGA implants were prepared using a new desktopmelt-processing system. EXPANSORB PLGA (PCAS, 50:50, MW-20,000 Da) andFITC-PLGA (Akina) were ground to a fine powder. The Qβ, HPV-Qβ, orCy5-Qβ particles were lyophilized before melt extrusion. The formulationof all implants was 80% PLGA, 10% VLP, and 10% PEG8000. The powderedcomponents were mixed in a 2-mL centrifuge tube by vortexing for 20 min,then loaded into the melt-processing system (maximum load =60 mg) andheated to 70° C. for 90 s. The air pressure was gradually increased upto 10 psi to extrude the implant cylinders, which were dried for 1 h andcut into 0.3-0.5 cm lengths, according to the weight.

Implant Analysis by SEM-EDX

Cross-sections of the implants were coated with carbon and observed byscanning electron microscopy with energy dispersive X-ray spectroscopy(SEM-EDX), using an FEI Quanta 600 scanning electron microscope coupledto a Bruker XFlash 6/60 EDX spectroscope. The EDX maps were preparedusing the FEI AZtec software.

Characterization of Particles

The VLPs were characterized by fast protein liquid chromatography(FPLC), transmission electron microscopy (TEM), dynamic light scattering(DLS), and sodium dodecylsulfate polyacrylamide gel electrophoresis(SDS-PAGE). FPLC was performed using an AKTA-FPLC 900 system fitted withSuperose 6 Increase 10/300 GL columns (GE Healthcare), using PBS (pH7.4) as the mobile phase at a flow rate of 0.5 mL/min. TEM images wereacquired on an FEI Tecnai Spirit G2 Bio TWIN transmission electronmicroscope. Samples were mounted on 400-mesh hexagonal copper grids andstained with 2% uranyl acetate. DLS was carried out on a MalvernInstruments Zetasizer Nano at 25° C., in plastic disposal cuvettes.SDS-PAGE was performed on NuPAGE 12% Bis-Tris protein gels (ThermoFisher Scientific) at 150 mV for 75 min. The gels were stained withCoomassie Brilliant Blue and images were acquired using theProteinSimple FluorChem R imaging system.

Analysis of the Loaded PLGA Implants In Vitro

Qβ-loaded PLGA implants (˜3 mg implant containing 300 μg VLPs) wereplaced in a 1.5-mL centrifuge tube and immersed in 500 μL PBS at 37° C.At each time-point, 200-_(i)lL samples were removed and replaced with200 μL fresh PBS. The quantity of released VLPs was determined bymeasuring the total protein concentration using a Pierce BCA assay kit(Thermo Fisher Scientific).

Analysis of Cy5-Qβ Loaded FITC-PLGA Implants In Vivo

All animal experiments were carried out in accordance withrecommendations from the UCSD's Institutional Animal Care and UseCommittee (ethical approval: UCSD IACUC protocol S18021). Female BALB/cmice aged 8 weeks (Jackson Laboratory, Bar Harbor, Me., USA) wereanesthetized for the implantation process before shaving hair from theback and wiping the skin with Alcohol Prep pads. The FITC-PLGA implantsloaded with Cy5-QP VLPs (˜1 mg implant containing 100 μg VLPs) wereintroduced subcutaneously via an 18-gauge needle by pushing the implantout of the needle with a sterilized stainless-steel wire (0.51 mmdiameter). The needle was withdrawn and the skin wiped with Alcohol Preppads. Fluorescence images were acquired on an IVIS 200 imaging system atdifferent time-points and were analyzed using Living Image v3.0.

Immunization

For vaccines in solution, female BALB/c mice aged 8 weeks werevaccinated subcutaneously by injection on days 0, 14, and 28, with bloodcollected 7 days after each vaccination. For the PLGA implants, micewere vaccinated by subcutaneous implantation, as described above, andblood was collected on days 7, 14, 21, 35, and 49. To compare thedifferent HPV-Qβ conjugates, n=5 mice were immunized three times with 30μg of the specific HPV-Qβ conjugate (or the Qβ as a control). For theconjugates vs. mixture vs. peptide experiment, n=6 mice were vaccinatedwith 30 μg of the specific HPV-Qβ conjugate, a mixture of 30 μg Qβ and 5μg of the HPV peptide, or 5 μg of the HPV peptide alone. For theevaluation of single-dose vaccination using the loaded PLGA implants,the PLGA implants were subcutaneously introduced into n=6 mice. Theimplants contained 100 μg HPV-Q13, 500 μg HPV-Qβ, a mixture of 100 μgQβ, and 20 μg HPV peptide, or 20 μg HPV peptide alone. To make a directcomparison among the three different vaccination techniques, threedifferent groups of mice each comprising n=6 mice were vaccinatedsubcutaneously with a single dose of 100 μg of the HPV-Qβ conjugate,three doses of 30 μg of the HPV-Qβ conjugate, and a single doseHPV-Qβ-loaded PLGA implants, loaded at 100 μg. Sera were separated fromthe collected blood through centrifugation at 2000x g for 20 min andwere stored at 4° C. (short term) or −80° C. (long term).

Serum Analysis and Determination of Antibody Titers

The abundance of antibodies against HPV peptides and Qβ particles inserum samples was determined by the enzyme-linked immunosorbent assay(ELISA). For the detection of a-HPV antibodies, 96-well Piercemaleimide-activated plates (Thermo Fisher Scientific) were coated with100 μL HPV peptides (10 μg/mL) per well in coating buffer (0.1 M sodiumphosphate, 0.15 M sodium chloride, 10 mM EDTA, pH 7.2). After overnightincubation at 4° C., the plates were washed three times with PBScontaining 0.1% Tween-20 (PBST) and then blocked with 150 μL cysteine(10 μg/mL) per well for 1 h, at room temperature. After three washes inPBST, serial dilutions of serum in PBST containing 1% bovine serumalbumin (BSA; Roche Diagnostics, Mannheim, Germany) were added andincubated at 37° C. for 1 h. After three washes in PBST, we added 100 μLof an alkaline phosphatase-labeled goat anti-mouse IgG secondaryantibody (Thermo Fisher Scientific) diluted 1:2000 in PB ST +1% BSA for30 min, at 37° C. Finally, the plates were washed five times with PBSTand developed with 100 μL of the 1-step PNP substrate (Thermo FisherScientific) for 30 min at 37° C. The reaction was stopped by adding 100μL of 1 M NaOH and the absorbance was read at 405 nm on a Tecanmicroplate reader. For the detection of a-Qβ antibodies, 96-wellPolySorp plates (Thermo Fisher Scientific) were coated with 100 μL Qβ(10 (Ig/mL in PBS) and incubated at 4° C. overnight. After three washesin PBST, the plates were blocked with 150 (IL PBST +2% BSA at 37° C. for1 h. The subsequent steps were as described for the detection of HPVpeptides.

The IgG subtype was also determined by ELISA. The procedure was similarto that described above for Qβ and HPV peptides, but the secondaryantibody was replaced with goat anti-mouse antibodies specific forIgG2a, IgG2b, and IgG1 (diluted 1:2000), as part of the IgG subtypeantibody kit (Sigma-Aldrich, St. Louis, Mo., USA). After three washes inPBST, we added an alkaline phosphatase-labeled rabbit anti-goat antibody(Sigma-Aldrich) diluted 1:3000, then washed five times in PB ST.Finally, the plates were developed by adding the 1-step PNP substrate,as described above. For all ELISAs, the antibody titers were defined asthe reciprocal serum dilution at which the absorbance exceeded thebackground value by >0.2.

Pseudovirus Production and Purification

HPV16 pseudovirus encapsidating the reporter plasmid pfwB (Addgene#37329) encoding green fluorescent protein (GFP) was produced in 293 TTcells, as previously described. In brief, 293 TT cells were transfectedwith a mixture of two plasmids—pfwB and p16Llw (Addgene #37320)containing the sequence of each HPV shell. After 48 h, the cells werelysed and mature pseudovirus was purified on an Optiprep gradient(27:33:39) through ultracentrifugation at 125,755×g for 6 h. Gradientfractions corresponding to the L1 band were collected and characterizedby SDS-PAGE, agarose gel electrophoresis, and flow cytometry, asrecommended. HPV16 pseudovirus stocks were titrated in pgsa-745 cells toyield 50-60% GFP-positive cells (percentage of infectivity) for theL2-based neutralization assay.

L2-Based Neutralization Assay

MCF10A cells were cultured in 96-well plates to produce theextracellular matrix (ECM), followed by the addition of the HPVpseudovirus (PsHPV), as previously described. The ECM-PsHPV wereincubated overnight, then removed and replaced with two-foldserial-dilutions of serum collected from immunized mice or the growthmedium, as a control. The plates were incubated for 6 h before addingpgsa-745 cells. After 48 h, HPV16 pseudovirus infectivity andneutralization were assessed by flow cytometry (Accuri) based on theexpression levels of GFP in infected cells. The reciprocal of thehighest serum dilution that inhibited 50% of the infection relative tothe control serum was considered the neutralization titer.

Statistical Analysis

Comparisons of the differences between two different groups wereperformed using unpaired two-tailed student's t-test (GraphPad Prismsoftware); * p<0.05; ** p<0.01; *** p<0.005; ns, not significantp >0.05. Values were expressed as means ±standard deviations. Samplesizes are stated in the figure legends.

Results and Discussion

The HPV16 epitope L217-31 is ideal for the development of an effectiveHPV vaccine because it is highly conserved among diverse HPV isolates.Different linkers and epitope orientations can have a significant effecton the immunogenicity of peptides, thus, we designed four peptidescontaining the L21₇₋₃₁ epitope, featuring alternative linkers (GPSL (SEQID NO: 10) or GGSGGGSG (SEQ ID NO: 6)) and orientations (epitope exposedat the N-terminus or C-terminus), as shown in Table 1. The peptides wereconjugated to the surface of the VLPs derived from bacteriophage Qβ in atwo-step procedure, where a bifunctional PEG was first conjugated viathe NHS-chemistry to surface amines of Qβ. Next, terminal cysteines fromthe HPV epitopes were conjugated via a maleimide linker to the PEG-Qβconjugate (FIG. 35A). SDS-PAGE analysis of the products confirmed thepresence of unmodified Qβ coat protein with a molecular weight of 14.2kDa and an additional band at -17 kDa, corresponding to the coat proteinconjugated to the HPV peptide (FIG. 35B). Unmodified and conjugated VLPsretained the dimeric assemblies under the electrophoresis conditions, asexpected due to the relative strength of the dimeric interaction. Therelative intensity of the bands indicated that -30% of the Qβ coatproteins were conjugated to HPV peptides (GPSL-N-expo 32.2%, GPSL-C-expo31.1%, GGSG-N-expo 33.8%, GGSG-C-expo 31.8%). FPLC analysis revealed asingle peak, indicating that the VLPs were monodisperse and did not formaggregates (FIG. 35C and FIG. 40). DLS revealed that the diameter of theVLPs was 28 ±3 nm, which was consistent with the TEM images (FIG. 35D,Eand FIG. 40). These results indicated that the conjugation of HPVpeptides did not alter the overall size or shape of the VLP asanticipated, given the low molecular weight of the peptides.

Mass (expected/ Purity Name Sequence observed) (%) GPSLQLYKTCKQAGTCPPDGPSLC 2110.5/2110.2 96.5 N-expo (SEQ ID NO: 11) GPSLCGPSLQLYKTCKQAGTCPPD 2110.5/2110.5 97.3 C-expo (SEQ ID NO: 12) GGSGQLYKTCKQAGTCPPDGGSGGG 2272.5/2272.3 96.4 N-expo SGC (SEQ ID NO: 13) GGSGCGGSGGGSGQLYKTCKQAGTC 2272.5/2272.2 95.3 C-expo PPD (SEQ ID NO: 14)

We found that all four conjugated VLPs (but not the unmodified Qβparticles) elicited the production of anti-HPV IgG in mice, but that thehigher titers were achieved when the epitope was exposed at theC-terminus (FIG. 35F). All four conjugated VLPs and unmodified Qβparticles generated similar IgG titers against the Qβ carrier, as wouldbe expected for a VLP (FIG. 35G). There was no significant difference intiter when comparing the two constructs with the HPV peptide exposed atthe C-terminus but at different linkers, even when using differentdilution factors for the second boost (FIG. 35H and FIG. 41).Ultimately, we selected the construct with the C-terminal epitope andthe GGSGGGSG (SEQ ID NO: 6) linker for the subsequent experiments andrefer to this construct hereafter as HPV-Qβ. The conjugation of HPV16epitope L21₇₋₃₁ to Qβ was necessary to enhance its immunogenicity,because the injection of a mixture of unmodified Qβ and the free HPVpeptide elicited antibodies against Qβ but not against HPV, and noantibodies against HPV were generated by the injection of the free HPVpeptide alone (FIG. 42).

HPV-Qβ was encapsulated into the PLGA implants through melt-extrusion,as previously reported. Parameters that were previously optimizedincluded temperature, such that the temperature was well above the T_(g)of PLGA, to allow for flow at a reasonably low pressure and residencetime in the extruder, to ensure complete melting of the polymer. Thismethod can be easily adapted to other vaccine candidates but requiresoptimization for each candidate to determine temperature stability,candidate release rate, etc. To optimize the control of the implantparameters, we used a benchtop melt-processing system (FIG. 36A). Takingadvantage of the stable extrusion pressure and adjustable nozzle size,the PLGA implants were produced with an accurate diameter of less than0.8 mm, so that they fit inside 18-gauge needles, for convenientadministration. The loaded PLGA implants were extruded as implantcylinders, 5-6 mm in length, which were cut into segments of 0.3-1 mmfor implantation (FIG. 36B). SEM imaging of implant cross-sectionsrevealed a uniform cylinder of -0.5 mm diameter (FIG. 36C). Homogeneousdispersion of HPV-Qβ particles was verified by the EDX analysis,specifically the sulfur K-series emission signal map (FIG. 36D). Ourprior experience indicates that homogeneous dispersion within theimplant improves the linearity of release and negates the aggregation ofVLPs from the released fraction.

The loaded PLGA implants were shown to slowly release the VLPs into PBS,with continuous release occurring over a period of up to 35 days at 37°C. (FIG. 37A). Released fractions indicated that the HPV peptidesremained conjugated following the melt-extrusion process, as confirmedby the presence of -17 kDa bands when the samples taken on days 15 and30 were analyzed by SDS-PAGE (FIG. 37B). TEM and DLS confirmed thepresence of intact HPV-Qβ particles with a diameter of -30 nm (FIGS.37C, D). FPLC analysis of the released HPV-Qβ particles showed that themelt-extrusion process did not promote aggregation either duringencapsulation or upon release (FIG. 37E).

The in vivo subcutaneous environment is more complex than PBS and mightinfluence the release of the cargo from the PLGA implants. We sought toevaluate the release kinetics of the implanted Qβ using fluorescencemolecular tomography in vivo. Unmodified Qβ was used for this set ofstudies, rather than HPV-Qβ due to the competing chemical reactions forsurface conjugation of fluorophores (i.e., HPV-Qβ exhausted the mostreactive amines for conjugation). To track the release of the particlesin vivo, Qβ was labeled with Cy5 and the incorporation of thefluorophore was confirmed by FPLC and SDS-PAGE (FIG. 43). FPLC analysisrevealed a peak in the 647 nm channel (Cy5 absorbance), at a retentionvolume of ˜11 mL, corresponding to the Qβ particles (FIG. 43A). SDS-PAGEanalysis showed a band corresponding to the anticipated molecular weightof Cy5-Qβ, with overlapping signals in the blue fluorescence andCoomassie Brilliant Blue channels (FIG. 43B).

To track the in vivo fate of the encapsulated VLPs and the polymericimplant, an FITC-PLGA matrix loaded with Cy5-Qβ particles was implantedsubcutaneously into mice and fluorescence images were captured atdifferent time-points (FIG. 38A). This allowed for separate imaging ofpolymer degradation and cargo release. The profiles of the Cy5 and GFPchannels are shown in FIG. 38B,C, respectively. A slight increase influorescence intensity was observed in both channels, immediately afterimplantation. The fluorescence intensity in both channels then declinedcontinually from day 3 to day 32, indicating the degradation of the PLGAand the slow release of the VLPs. The degradation of PLGA was closelycorrelated to the decline of the VLP signal. This experiment confirmedthat the PLGA implants produced by melt-extrusion retained their abilityto release cargo slowly over time in vivo and that degradation kineticsof the polymer play the main role in cargo release.

To assess the efficacy of the vaccine candidate delivered using thevaccine delivery device, we subcutaneously vaccinated the mice with PLGAimplants loaded with HPV-Qβ at two different doses (100 μg and 500 μg).As controls, we vaccinated mice with PLGA implants loaded with a mixtureof the HPV peptide and unmodified Qβ, or with the HPV peptide alone.Finally, we also subcutaneously immunized mice with three doses of freeHPV-Qβ (30 μg per dose), as a conventional vaccination control. Theimmunization and bleeding schedule is summarized in FIG. 39A.

We compared the anti-HPV IgG titers in serum samples taken from mice ineach of the experimental and control groups (FIG. 39B). The PLGAimplants loaded with HPV-Qβ elicited an intense immune response, withthe high HPV-specific IgG titers starting from day 14 and lasting to day49. There was no significant difference in the IgG titers between thegroups receiving 100 μg and 500 μg HPV-Qβ in the implant, indicatingthat 100 μg HPV-Qβ is an adequate quantity of the antigen forimmunization. The high titers elicited by the single-dose HPV vaccineimplant were maintained over 49 days and were similar to the titerselicited by the prime, first boost, and second boost schedule, with freeVLPs. The PLGA implants loaded with the HPV peptide or a mixture of theHPV peptide and unmodified Qβ particles did not elicit a significantimmune response against HPV, demonstrating that the PLGA matrix had noimpact on the immunogenicity of the antigens but only ensured their slowrelease (FIG. 39B). The anti-Qβ IgG titer was consistent with thatagainst the HPV peptide. High IgG titers against Qβ were detected fromday 14 to day 49 in mice vaccinated with PLGA implants containing HPV-Qβor the mixture of unmodified Qβ and the HPV peptide (FIG. 44A).

The IgG subtype ratio following vaccination indicates the mechanism ofthe immune response. Mice vaccinated with the single-dose HPV-Qβ vaccinebased on the PLGA implant, showed a similar HPV-specific IgG subtyperatio to mice in the conventional vaccination control group (FIG. 39C).In both cases, IgG2a was the predominant subtype (˜50%), followed byIgG1 (˜40%) and IgG2b (˜7%). Interestingly, the Qβ-specific IgG subtyperatio was distinct, with IgG2a accounting for up to 70% and IgG1 only15% of the total IgG titer in both single-dose vaccine group and theconventional vaccination control group (FIG. 44B). This indicates thatthe cleavage of the bond between the HPV epitope and Qβ carrier is aspecific step during immune response, with the separate components thenfollowing different immune response pathways.

Additionally, we aimed to evaluate to compare a single-dose solubleinjection versus a single-dose biodegradable implant, in an effort toevaluate the necessity for a prime-boost regime with this vaccinecandidate. Three groups were studied—(1) a single dose of biodegradable100m HPV-Qβ loaded PLGA implant, (2) a single dose of subcutaneousinjection of 100m of HPV-Qβ solution, and (3) three subcutaneousinjections of 30 μg HPV-Qβ solution. The vaccination and bleedingschedule are shown in FIG. 45A. Data indicate comparable levels ofHPV-specific IgG antibody titers independent of the schedule (primeboost vs. single administration) or delivery strategy (injection vs.implant), throughout the entire time of observation for up to 90 days(FIGS. 45B, C). This indicates that this particular HPV-Qβ vaccinecandidate might be effective after single dosing.

We find that in the case of slow release from the implant, the IgGantibody titer was slightly lower after 14 days and reached the samelevel as obtained by the other methods after 28 days. In all cases, theantibodies remained similar even after three months. Since the lifetimeof HPV antibodies in mouse is found to be much longer (>90 days) thanthe release time of the vaccine cargo from the implant (about 20-25days), a simple model of exponential loss of antibody in the mouse andan approximately linear release of vaccine cargo from the implantsupports our observations. The model demonstrates that decay kineticswould dominate the concentration of antibodies in blood in the long runand would be essentially same for all methods of administration for thisvaccine. Hence, we conclude that the mice vaccinated with theslow-release implants obtain, after a reasonable time, IgG titerscomparable to that obtained by the traditional one single full dose orthree-dose strategy. While this result might be disappointing, we wouldlike to point out that subcutaneous injections are very expensive andrequire trained healthcare professionals, thus limiting its availabilityin developing and poor countries. Furthermore, we speculate that at alower dose, the implant group might show some advantages of the singledose injection; and this will be tested in future work. If this holdstrue; this would reduce production costs, which would also be ofbenefit. Nevertheless, the implications of melt manufacturing ofimplantable devices have the potential to be immense. Microneedlepatches can be manufactured through injection molding, which has theadvantage of massive scalability. These devices show similar efficacywhen self-administered, and given the known stability of Qβ, mighteliminate cold-chain requirements. We anticipate that this techniquewould be of great value for vaccines that require a prime-boost dosingregimen. Additionally, several recent studies also point out thebenefits of sustained release of antigen over bolus injection in termsof better antigen recognition features and enhancement of germinalcenter responses.

In order to demonstrate the efficacy of the single-dose HPV-Qβ vaccine,serum antibodies from the vaccinated mice were tested for their abilityto prevent the infection of pgsa-745 cells by HPV pseudovirus in an invitro neutralization assay (FIG. 46), as previously reported for othervaccine candidates. Data indicate that antibodies raised were indeedneutralizing. The relative number of infected GFP-positive cells wassignificantly lower when the serum from animals immunized with HPV-Qβ(100 or 500 μg) was added during the assay, compared to serum fromanimals vaccinated with a mixture of unmodified Qβ and the HPV peptide,or the HPV peptide alone (FIG. 39D). The neutralization titers for eachtreatment were 1:750 for HPV-Qβ (100 μg), 1:250 for HPV-Qβ (500 μg), andeffectively zero neutralizing activity for the free peptide, with orwithout unmodified Qβ.

Our results demonstrate the feasibility and efficacy of a single-doseHPV vaccine based on a biodegradable polymer implant. This is of highimportance for future development of self-administered vaccine devicesmade by polymer melt processing. It is also important to note that thehigh temperature stability of the HPV-Qβ vaccine candidate serves twofunctions—(1) it allows for traditional polymer manufacturing methods toscale-up fabrication of the implant, and (2) the vaccine and its implantwould eliminate the need for the cold chain, thus enabling globaldistribution. The design concepts are highly adaptable and would beapplicable to any infectious disease. The concepts described areespecially appealing because they are scalable and fit within thetraditional workflow of pharmaceutical production. Bacterialfermentation is a common method to generate therapeutic proteins andVLPs provide an especially high yielding platform with minimaloptimization (Qβ evolved to be expressed in bacteria). The vaccineadministration described herein mimics that of the clinically approveddevice, Nexplanon. Finally, since the VLP serves as a carrier, newepitopes can be quickly adapted to new diseases or strains.

From the above description of the invention, those skilled in the artwill perceive improvements, changes and modifications. Suchimprovements, changes and modifications within the skill of the art areintended to be covered by the appended claims. All references,publications, and patents cited in the present application are hereinincorporated by reference in their entirety.

Having described the invention, we claim:
 1. A construct comprising: amelt processed blend of a biodegradable polymer and viral nanoparticles,wherein the viral nanoparticles are encapsulated within a biodegradablepolymer matrix of the biodegradable polymer, the viral nanoparticlesincluding a plurality of immunogenic peptides conjugated to the viralnanoparticles, the construct upon administration to a subject providingsustained release of the viral nanoparticles and conjugated immunogenicpeptides from the matrix to the subject, the released viralnanoparticles and conjugated immunogenic peptides eliciting animmunogenic response in the subject.
 2. The construct of claim 1,wherein the conjugated immunogenic peptides upon release from thepolymer matrix eliciting substantially the same or enhanced immunogenicresponse as immunogenic peptides conjugated to viral nanoparticles notencapsulated within the biodegradable polymer.
 3. The construct of claim1, wherein the plurality of immunogenic peptides are conjugated to outersurfaces of the viral nanoparticles with linkers.
 4. The construct ofclaim 3, wherein the linker is heterobifunctional linker.
 5. Theconstruct of claim 1, wherein the viral nanoparticles are substantiallyuniformly dispersed in the polymer matrix.
 6. The construct of claim 1,wherein the biodegradable polymer material has a melt temperature belowthe degradation temperature of the viral nanoparticles.
 7. The constructof claim 5, wherein the biodegradable polymer material comprisespoly(lactic-co-glycolic acid) (PLGA) or a copolymer thereof andoptionally at least one porogen.
 8. The construct of claim 1, whereinthe viral nanoparticles include bacteriophages, plant viruses, and/orplant virus-like particles.
 9. The construct of claim 1, the viralnanoparticles having a release profile from the biodegradable polymermatrix at least partially defined by the degradation of thebiodegradable polymer material under physiological conditions.
 10. Theconstruct of claim 1, including up to about 25% by weight of the viralnanoparticles.
 11. The construct of claim 1, wherein the melt processingof biodegradable polymer and the viral nanoparticles is at a Pecletnumber of about 5 to about
 25. 12. The construct of claim 1, beingconfigured as a plurality of microparticles or microneedles.
 13. Asingle dose vaccine implant comprising: a biodegradable polymer; and aplurality of viral nanoparticles melt processed with and encapsulatedwithin a biodegradable polymer matrix that includes the biodegradablepolymer, the viral nanoparticles including a plurality of exogenousimmunogenic peptides conjugated to the viral nanoparticles, the implantupon administration to a subject providing sustained release of theviral nanoparticles and conjugated immunogenic peptides from the matrixto the subject, the released viral nanoparticles and conjugatedimmunogenic peptides eliciting an immunogenic response in the subject.14. The implant of claim 13, wherein the conjugated immunogenic peptidesupon release from the polymer matrix eliciting substantially the same orenhanced immunogenic response as immunogenic peptides conjugated toviral nanoparticles not encapsulated within the biodegradable polymer.15. The implant of claim 13, wherein the plurality of immunogenicpeptides are conjugated to outer surfaces of the viral nanoparticleswith heterobifunctional linkers.
 16. The implant of claim 13, whereinthe viral nanoparticles are substantially uniformly dispersed in thepolymer matrix.
 17. The implant of claim 13, wherein the biodegradablepolymer material has a melt temperature below the degradationtemperature of the viral nanoparticles and immunogenic peptide.
 18. Theimplant of claim 13, wherein the biodegradable polymer materialcomprises poly(lactic-co-glycolic acid) (PLGA) or a copolymer thereofand optionally at least one porogen.
 19. The construct of claim 13,wherein the viral nanoparticles include bacteriophages, plant viruses,and/or plant virus-like particles.
 20. The implant of claim 13, theviral nanoparticles having a release profile from the biodegradablepolymer matrix at least partially defined by the degradation of thebiodegradable polymer material under physiological conditions.
 21. Theimplant of claim 13, including up to about 25% by weight of the viralnanoparticles.
 22. The implant of claim 13, being configured as aplurality of microparticles or microneedles.